BIOLOGY 

J 
G 


PRACTICAL    PHYSIOLOGICAL    CHEMISTRY. 


LTD] 


LONDON    AGENTS: 
SIMPKIN,    MARSHALL    6?    Co.    LTD. 


Practical  Physiological 
Chemistry- 


SYDNEY   W.    COLE,   M.A. 

Trinity  College,   Cambridge, 
Demonstrator  of  Physiology,  Cambridge  University. 


FOURTH    EDITION 


CAMBRIDGE  : 
W.    HEFFER    &    SONS    LTD. 

1914 


a 

°%4 
^n 

BlOLOSf 


PREFACE    TO    THE    FIRST    EDITION, 


MY  aim  in  writing  this  book  has  been  to  present  to  the  student  a 
series  of  exercises  which  can  be  successfully  carried  through  in 
ordinary  class  work. 

Too  often  a  student  is  discouraged  in  his  work  and  displeased 
with  his  Text- Book  by  finding  that  a  minute  care  in  following  the 
instruction  given  fails  to  produce  the  specified  result.  I  trust  that 
no  such  difficulty  will  be  met  with  in  working  through  this  Book. 
Each  and  every  exercise  given  here  I  have  first  worked  through 
and  obtained  the  result  stated.  All  I  ask  of  the  student  is  a  zealous 
and  interested  care  and  he  will  then  have  no  difficulty  in  performing 
the  experiments  and  learning  the  lessons  they  teach. 

The  ground  covered  is  more  than  is  at  present  necessary  for 
most  examinations  in  medicine,  but  I  feel  that  this  is  justified  by 
the  growing  importance  of  the  subject  and  the  increasing  standard 
of  the  knowledge  of  it  required  of  candidates  at  these  examinations. 

A  special  feature  of  the  book  is  the  notes  that  follow  certain  of 
the  exercises.  These  notes  summarise  a  series  of  exercises, 
indicate  the  special  precautions  that  are  necessary  for  success  or 
give  the  probable  reasons  for  an  apparent  failure  in  the  performance 
of  a  given  exercise.  They  should  be  carefully  studied  both  before 
and  after  the  exercise  to  which  they  refer.  At  the  end  of  the  book 
spaces  are  provided  for  the  student  to  draw  various  crystalline  forms 
from  preparations  made  by  himself.  I  consider  this  a  more  in- 
structive plan  than  giving  illustrations  of  typical  crystals,  which 
often  differ  considerably  from  those  prepared  in  class  work.  A 
blank  chart  for  recording  the  absorption  spectra  of  various  pigment 
solutions  and  colour  reactions  is  also  added.  The  drawings  should 
be  shown  to  the  demonstrator  of  the  class  for  comments  or 
corrections. 

SYDNEY    W.    COLE. 
TRINITY    COLLEGE, 
CAMBRIDGE. 

November,  1904. 


304482 


PREFACE    TO    THE    THIRD    EDITION. 


THE    present    volume    is    an    outcome    of    the    two    editions    of 
the  Author's  "  Practical  Exercises  in  Physiological  Chemistry." 

The  increasing  importance  of  the  science  to  medical  men  has 
created  a  demand  for  a  book  that  embodies  precise  instruction 
for  class-work  with  an  account  of  the  properties  and  significance 
of  the  more  important  physiological  substances.  The  present 
work  is  an  attempt  to  realise  these  desiderata. 

The  Author  wishes  to  draw  particular  attention  to  the 
analytical  methods.  It  is  lamentable  that  for  the  investigation 
of  the  nitrogenous  excretion  of  a  patient,  the  average  medical 
man  has  at  present  only  one  method  at  his  command.  That 
method,  the  hypobromite,  is  notoriously  unreliable,  and  the 
conclusions  drawn  from  it  may  be  extremely  misleading.  It  is 
sincerely  hoped  that  all  medical  students  will  be  taught  the 
microchemical  methods  of  urinary  analysis  introduced  by  Folin. 
The  Author  is  convinced  that  they  are  reliable,  and  that  the 
average  medical  man  could  conduct  them  rapidly  with  a  very 
small  amount  of  special  apparatus.  If  such  training  were  uni- 
versally adopted  in  England,  an  enormous  amount  of  clinical 
material  that  is  now  wasted  would  become  available  for  research, 
and  a  rapid  increase  in  our  knowledge  of  physiology  and  pathology 
would  inevitably  follow. 

The  qualitative  methods  for  urinary  analysis  also  have  been 
considerably  modified  in  recent  years,  especially  in  regard  to 
sugar.  Fehling's  method,  that  has  for  so  long  been  the  crucial 
test,  is  unreliable.  It  should  be  supplanted  as  soon  as  possible 
by  more  conclusive  methods,  such  as  those  described  in  the 
section  on  glucose  in  urine. 

By  a  judicious  selection  of  exercises  the  book  can  be  adapted 
for  elementary  or  advanced  classes. 


PREFACE.  vii. 

The  Author  gratefully  acknowledges  his  indebtedness  to  Mr. 
H.  M.  Spiers,  of  Caius  College,  for  invaluable  help  in  reading 
the  proofs,  and  to  Messrs.  J.  Griffin  &  Sons  and  Messrs. 
Baird  &  Tatlock  for  the  loan  of  certain  of  the  diagrams. 

SYDNEY  W.  COLE. 
TRINITY   COLLEGE, 
CAMBRIDGE, 

April,  1913 


PREFACE    TO    THE    FOURTH    EDITION. 


LITTLE  more  than  a  year  has  elapsed  since  the  third  edition 
of  this  book  was  issued,  but  my  publishers  inform  me  that  the 
edition  is  exhausted.  I  venture  to  hope  that  this  rapid  sale 
indicates  that  both  medical  students  and  practitioners  are 
realising  the  immense  importance  of  accurate  analytical  methods, 
and  that  the  old  so-called  clinical  methods  are  essentially  of  the 
past. 

In  order  that  the  third  edition  may  not  be  rendered 
obsolete  and  unusable  by  the  issue  of  this  edition  I  have  not 
altered  the  arrangement  of  the  book  but  I  have  enclosed  in 
an  appendix  a  number  of  new  or  improved  methods.  In 
particular  I  would  draw  attention  to  the  recent  methods  for  the 
microchemical  analysis  of  sugar  in  blood.  Bang's  method  can 
be  performed  on  about  three  drops  of  blood  drawn  from  a 
finger  prick,  and  I  have  completely  satisfied  myself  of  its 
accuracy.  Its  extended  use  may  do  something  towards  the 
elucidation  of  the  mystery  of  diabetes.  Improved  methods  are 
given  for  the  analysis  of  glucose  and  lactose  in  pure  solutions, 
for  the  microchemical  analysis  of  uric  acid  in  urine,  for  chlorides 
in  blood  and  urine,  and  for  acetone  and  aceto-acetic  acid.  I 
have  also  added  two  recent  tests  of  my  own  for  minute  amounts 
of  glucose  in  urine  and  for  bile  pigments.  Finally  I  have 


Vlll.  PREFACE. 

included  Wohlgemuth's  method  for  the  estimation  of  urinary 
diastase  which  has  proved  of  such  service  in  the  diagnosis  of 
pancreatitis. 

I  have  to  thank  Professor  Langley  for  the  loan  of  Fig.  10, 
and  Messrs.  J.  J.  Griffin  and  Sons  for  Figs.  4,  5,  11,  12  and  13. 

In  conclusion,  I  desire  to  thank  the  many  teachers  who 
have  sent  me  letters  of  advice,  friendly  criticism  and  encourage- 
ment. If  this  book  has  helped  to  further  the  study  of  analytical 
methods  in  a  number  of  physiological  schools  both  in  England 
and  abroad  I  shall  feel  that  one  piece  of  good  work  has  been 
accomplished. 

SYDNEY    W.    COLE. 
CAMBRIDGE, 

May,  1914. 


CONTENTS. 


CHAPTER  I. 

PAGE. 

The  Proteins      ...         ...         ...         ...         ...  1 

A.  Classification...          ...          ...          ...  1 

B.  General  Properties ...          2 

C.  Colour  Reactions       ...         ...         ...  3 

D.  Albumins  and  Globulins  of  Serum 6 

E.  The  Chemistry  of  Egg-white           15 

F.  The  Gluco-proteins 17 

G.  The  Nucleoproteins  and  Nucleohistones 18 

H.     The  Metaproteins 22 

I.       The  Albumoses  and  Peptones         ...          ...  23 

J.       The  Scleroproteins _  28 

CHAPTER  II. 

The  Carbohydrates       ...          ...          ...          ...  31 

A.  The  Monosaccharides            ...          ...          ...  31 

B.  The  Disaccharides 38 

C.  The  Polysaccharides...          ...          ...  42 

D.  The  Quantitative  Estimation  of  Sugar      51 

CHAPTER  III 

The  Fats  and  their  Decomposition  Products  59 


X.  CONTENTS. 

CHAPTER  IV. 

PAGE. 

The  Chemistry  of  Some  Foods            67 

A.  Milk 67 

B.  The  Clotting  of  Milk             ...  69 

C.  Cheese             ...         ...          ...         ...         ...         ...  71 

D.  Potatoes         71 

E.  Flour 72 

F.  Bread... 73 

G.  Muscle  (Meat)           74 

CHAPTER  V. 

The  Composition  of  the  Digestive  Juices  and  the  Action  of 

Certain  Enzymes       82 

A.  Saliva 84 

B.  Pepsin             88 

C.  The  Acidity  of  Gastric  Juice            91 

D.  Trypsin           94 

CHAPTER  VI.       » 

The  Coagulation  of  Blood        99 


CHAPTER  VII. 

The  Red  Blood  Corpuscles  and  the  Blood  Pigments  ...  103 

A.  The  Laking  of  Blood  103 

B.  Haemoglobin  and  its  Derivatives   ...          ...          ...  105 

C.  The    Spectroscopic    Examination    of    the    Blood 

Pigments  ...         ...         ...         ...         ...       107 

CHAPTER   VIII. 
The  Constituents  of  Bile  115 


CONTENTS,  XI. 

CHAPTER  IX. 

PAGE. 

Urine  and  its  Chief  Constituents         123 

A.  The  Average  Composition 123 

B.  The  Physical  Chemistry  of  the  Urine        124 

I.  General  Properties  ...         ...         ...          ...  124 

II.  The  Specific  Gravity            124 

III.  The  Osmotic  Pressure  (Cryoscopy)            ...  126 

IV.  Acidity           129 

C.  The  Pigments  of  Urine        131 

D.  The  Inorganic  Constituents...          133 

E.  Urea 139 

F.  Uric  Acid       144 

G.  Purine  Bases  other  than  Uric  Acid             ...         ...  151 

H.    Creatinine  and  Creatine        152 

I.      Ammonia        ...          ...         ...         ...          ...          ...  153 

J.       Hippuric  Acid            154 

K.     Certain  Constituents  of  Abnormal  Urine  ...         ...  155 

1.  Albumin  and  Globulin           155 

2.  Albumoses       ...         ...          ...          ...          ...  156 

3.  Bence-Jones'  Protein...         ...         ...         ...  157 

4.  Blood  Pigments ' 158 

5.  Bile      159 

6.  Glucose            160 

7.  Fructose  (laevlose) 163 

8.  Pentose            163 

9.  Lactose            164 

10.  The  Acetone  Bodies 165 

11.  Glycuronic  Acid         ...          ...         ...         ...  167 

12.  Indican            168 

L.     Urinary  Sediments    ...          ...          ...          ...          ...  169 

CHAPTER  X. 

The  Quantitative  Analysis  of  Urine  ...           171 

Total  Nitrogen  (Kjeldahl)            173 

Total  Nitrogen  (Microchemical) 175 


xii.  CONTENTS. 

PAGE. 

Ammonia  (Folin)  ...          ...          ...          ...          ...          ...  179 

Ammonia  (Microchemical)           ...          ...          ...          ...  180 

Ammonia  (Formaldehyde)            ...          ...          ...          ...  181 

Urea  (Benedict) ...         181 

Urea  (Microchemical)       183 

Urea  (Hypobromite)         185 

Uric  Acid  (Folin-Schaffer)           ...          188 

Uric  Acid  (Microchemical)           189 

Creatinine  (Folin) 191 

Titration  Acidity  (Folin) ^-£  1 93 

Chlorides  (Volhard) ~T7  194 

Phosphates  (Uranium)      ...          ...          ...          ...          ...  196 

Total  Sulphates  (Folin) '       197 

Inorganic  Sulphates  (Folin)         198 

Ethereal  Sulphates            ...         198 

Total  Sulphur  (Benedict) ...  198 

Albumin  (Esbach) ' 199 

Albumin  (Scherer) ..."       199 

CHAPTER  XL 

The  Detection  of  Substances  of  Physiological  Interest       ...  200 

A.  Fluids 200 

B.  Solids ...  209 

APPENDIX. 

Weights  and  Measures...                                                          ...  211 

Tension  of  Aqueous  Vapour    ...                                              ...  2 1 2 

Atomic  Weights            ...  212 

Preparation  of  Normal  Solutions  of  Acids  and  Alkalies      ...  213 

Charts  for  Recording  Crystalline  forms         215 

Chart  for  Recording  Spectroscopic  Absorption  Bands         ...  222 

RECENT  METHODS. 

The  Estimation  of  Glucose  by  Bang's  Method  II 225 

The  Estimation  of  Glucose  by  Peters'  Method         227 


CONTENTS.  xiii. 

PAGE. 

The  Estimation  of  Lactose  by  the  Copper-iodide  Method  ...  231 

The  Micro-analysis  of  Sugar  in  Blood  by  Bang's  Method  ...  233 

The  Micro-analysis  of  Chlorides  in  Blood  by  Bang's  Method  237 

The  Estimation  of  Chlorides  in  Urine  by  Larrson's  Method  240 

The  Estimation  of  the  Acetone  Bodies  in  Urine       241 

The  Microchemical  Estimation  of  Uric  Acid            244 

The  Estimation  of  the  Diastatic  Power  of  Urine     246 

The  Estimation  of  Pepsin  by  Fuld's  Method            248 

Cole's  Test  for  Glucose  in  Urine         249 

The  Detection  of  Acetone  and  Aceto-acetic  Acid      ...         ...  250 

Cole's  Test  for  Bile  Pigments '.         251 

Preparation  of  Haemin  Crystals         252 


Logarithm  Tables         On  back  cover 


LIST    OF    ILLUSTRATIONS. 


Fig.  PAGE. 

1.  Apparatus  for  Sugar  Estimation  ...          ...  53 

2.  Zeiss'  Direct-vision  Spectroscope          107 

3.  Urinometer            125 

4.  Beckmann's  Freezing  Point  Apparatus  ...      "  ;^*^    128 

5.  Beckmann's  Thermometer           ...  "...  128 

6.  Folin's  Fume-absorber 172 

7.  Apparatus  for  Kjeldahl's  Method           174 

8.  Apparatus  for  Folin's  Microchemical  Methods  ...  176 

9.  Folin's  Apparatus  for  Estimation  of  Ammonia  ...  179 

10.  Apparatus  for   Urea    Determination  by   Hypobromite 

Method           ..<»*  186 

11.  Dubosq's  Colorimeter      ...  19* 

12.  Path  of  Ray's  in  Dubosq's  Colorimeter            192 

13.  Esbach's  Albuminometer             199 

14.  Flask  Fitted  for  Sugar  Estimation        ...  226 

15.  Apparatus  for  Maintaining  a  Standard  Heating  Power  229 

16.  Filtering  Apparatus  for  Reduced  Colour  ...          ...  230 

17.  Curve  Showing  Amount  of  Copper  Reduced  by  Glucose  232 

18.  Curve  Showing  Amount  of  Copper  Reduced  by  Lactose  233 

19.  Apparatus  for  Titration  in  an  Atmosphere  of  CO2       ...  236 

20.  Gooch  Crucible  and  Filtering  Apparatus          239 

21.  Apparatus  for  the  Estimation  of  Acetone         ...          ...  243 


ALTERATIONS,  CORRECTIONS  AND  OMISSIONS. 


The  reader  is  advised  to  make  the  necessary  corrections  without 
delay. 

p.  33,  1.  2.     For  "  lactore  "  read  "lactone." 
p.  46,  1.  16.     For  "  Achrodextrin  "  read  "Achroodextrin." 
p.  153,    Ex.  273.      For    "To    the    yellow"    read    "Heat   the    yellow." 
Insert  a  comma  after  "exercise." 

p.  162,  Ex.  293.     For  "5  or  6"  read  "10." 

p.  164,  Ex.  298.  Bial's  reagent  consists  of  1  to  1'5  gm.  orcine,  500  c.c. 
of  concentrated  hydrochloric  acid  and  30  drops  of  a  1  p.c. 
solution  of  ferric  chloride. 

p.  174,  1.  6  from  bottom.     For  "35"  read  "50." 
p.  177,  1.  10.     .For"3"  read  "4." 

p.  177,  1.  21  to  23.  Delete  "  To  each  flask  .  .  .  Nessler'  s  solu- 
tion." 

p.  177,  1.  3  from  bottom.     For  "20"  read  "10." 
p.  184,  1.  13.     For  "179"  read  "177." 
p.  185,  1.  3.     For  "  0'45  "  read  "0'045." 

p.  190.  Preparation  of  a  stable  solution  of  uric  acid.  Dissolve  1  gm. 
of  uric  acid  in  200  c.c.  of  0'4  p.c.  lithium  carbonate.  Add 
40  c.c.  of  40  p.c.  formaldehyde.  Shake  and  allow  to  stand  a 
few  minutes.  Add  20  c.c.  of  normal  acetic  acid.  Make  up  to 
1  litre  with  water.  Standardize  colorimetrically  the  next  day 
against  a  freshly  prepared  solution  of  uric  acid  (see  p.  ]90), 
using  5  c.c.  of  the  formalin-uric-acid  solution.  This  should 
contain  very  nearly  1  mg.  of  uric  acid  that  reacts  with  Folin's 
reagent.  The  solution  is  quite  stable. 

p.  198,  Ex.  322.     Benedict's  sulphur  reagent  is— 
Crystallised  copper  nitrate,  200  gm. 
Potassium  chlorate,  50  gm. 
Distilled  water  to  1  litre. 

p.  205,  1.  11  from  bottom.     For  "249"  read  "248." 


CHAPTER   I. 

THE    PROTEINS. 

These  bodies  are  composed  of  certain    amino-acids 
and  bases  condensed  in  varying  proportions. 

A.    Classification. 

1.  Protamines.      Basic   substances,  containing  a  high   per- 
centage of  nitrogen  and  formed  almost  entirely  of  bases.     They  are 
found  in  ripe  spermatozoa  and  ova.     They  form  salts  with  acids. 

2.  Histones.     Similar  to   the  protamines,    but    less   rich    in 
nitrogen  and   bases.     Found  in  unripe  spermatozoa,  the  stroma  of 
red    corpuscles,    and    in    the    thymus.     They   are    precipitated    by 
ammonia. 

3.  Globulins  insoluble  in  water  ] 

f  coagulated  by  boiling. 

4.  Albumins  soluble  in  water   J 

5.  Glutelins.     Insoluble  in  water  and  alcohol :  \ 

soluble  in  dilute  acid  or  alkali  !    Found  in 

6.  Gliadins.     Insoluble    in   water :    soluble  in  j      cereals. 
75  %  alcohol 

7.  Sclero-proteins.       Forming    the    skeletal    structure    of 
animals  ;  e.g.  keratin,  elastin,  collagen  (the  anhydride  of  gelatin). 

8.  Phospho-proteins.       Proteins    rich    in   phosphorus,    e.g. 
caseinogen  of  milk  and  vitellin  of  egg-yolk. 

9.  Conjugated-proteins.     Proteins  joined  to  a  non-protein 
("  prosthetic  ")  group. 

(i)     Chromoproteins.  Protein  +  pigment     molecule,    e.g. 
haemoglobin. 

(ii)     Nucleoproteins.  Protein  +  nuclein  or  nucleic  acid, 

(iii)     Glucoproteins.  Protein  +  carbohydrate,  e.g.  mucin. 


THE     PROTEINS. 


[CH.    T. 


10.     Hydrolysed   Proteins.     Proteins  formed  by   the  action 
of  acids,  alkalies  and  certain  enzymes,  on  native  proteins, 
(i)     Metaproteins. 
(ii)     Albumoses  or  Proteoses. 
(iii)     Peptones. 

(iv)     Polypeptides — formed  of  amino-acids  only. 
B.     General   Properties. 

1.  Certain    colour    reactions    are    given    dependent    on    the 
existence  of  certain  constituent  radicles.     (See  Exs.  1-6.) 

2.  They  are  precipitated  by  the  so-called  alkaloidal  reagents 
(Ex,  14-18). 

3.  They    are    precipitated    by    salts    of    the    heavy    metals 
(Ex.  19-22). 

4.  They   are   colloidal.     That  is,  they  do  not  diffuse  through 
animal    membranes,    and    the    large   molecules  tend    to    aggregate 
together  under  the  influence  of  heat,  neutral  salts,  etc.,  to  form  a 
precipitate  or  coagulum. 

Solubilities  of  the  chief  proteins. 

S  =  Soluble.     I  =  Insoluble. 


Water. 

Dilute 
Salts. 

Dilute    i     Dilute        i-safd. 
Acetic    i  Alkalies  |  Am  so  . 
Acid. 

Sat'd. 
Am-2S04. 

Globulin  -         -         -         - 

I 

S 

S                S 

I 

I 

Albumin  - 

S 

S 

S                 S 

p 

I 

Metaprotein 

I 

I 

S                 S 

I 

I 

Primary  Albumose  -         -         S               S 

S              S 

I 

I 

Secondary  Albumose        -  I       S               S 

S              S 

S 

I 

Peptone  - 

S 

S 

S             S 

S 

S 

Caseinogen 

I          I 

I              S 

1 

I 

Nucleoprotein  - 

I          I 

I              S 

I 

I 

Mucin      ---         - 

I 

I 

I              S 

I 

I 

Gelatin    - 

S* 

S* 

S*            S* 

I 

I 

Keratin    -                                       I 

I 

I          I 

I 

I 

*  If  warmed. 


CH.    I.]  COLOUR     REACTIONS.  3 

C.     The   Colour   Reactions   of  Proteins. 

For  t'he  following  reactions  use  egg-white  that  has  been  well  beaten  with 
six  times  its  volume  of  water,  or  serum  that  has  been  diluted  ten  times  with 
water. 

1.  The  Xanthoproteic  reaction.  To  5  c.c.  of  the  protein 
solution  in  a  test-tube  add  about  one-third  of  its  volume  of  strong 
nitric  acid.  A  white  precipitate  is  formed.  Boil  for  a  minute. 
The  precipitate  turns  yellow  and  partly  dissolves  to  give  a  yellow 
solution.  Cool  under  the  tap  and  add  strong  ammonia  till  the 
reaction  is  alkaline.  The  yellow  colour  is  turned  to  orange. 

NOTES. — 1.  The  essential  features  of  the  reaction  are  that  a  yellow  colour 
is  obtained  when  the  solution  is  boiled  with  strong  nitric  acid,  and  that  this 
yellow  colour  is  intensified  on  the  subsequent  addition  of  ammonia. 

2.  The  precipitate  is  due  to  the  formation  of  metaprotein  by  the  action 
of  nitric  acid   on   albumins  or  globulins,   this  metaprotein  being  insoluble  in 
strong  mineral  acids.    (See  Ex.  13.)    Itfollowsthatalbumoses  and  peptones,  etc., 
do  not  give  the  precipitate  with  nitric  acid. 

3.  The  yellow  colour  is  due  to  the  formation  of  a  nitre-compound  of  some 
aromatic  substance,  i.e.  a  substance  containing  the  benzene  ring. 

4.  The  aromatic  substances  in  the  protein  molecule  that  are  responsible 
for  the  reaction  are  tyrosine,  tryptophane  and  phenyl  alanine. 

5.  Oleic  acid,  olive  oil  and  most  vegetable  oils  give  a  well-marked  xantho- 
proteic' reaction. 

2.  Millon's  reaction.  Treat  5  c.c.  of  the  protein  solution 
with  half  its  volume  of  Millon's  reagent.  A  white  precipitate  is 
formed.  Boil  the  mixture.  The  precipitate  turns  brick-red  in 
colour,  or  disappears  and  leaves  a  red  solution. 

NOTES. — 1.  The  essential  feature  of  the  reaction  is  the  red  colour  on 
boiling.  The  white  precipitate  in  the  cold  is  due  to  the  action  of  the  mercuric 
nitrate  on  the  proteins.  (See  Ex.  19.) 

2.  A  white  precipitate  is  also  obtained  with  solutions  of  urea.  (See  Ex.  251.) 

3.  Sulphates  give  a  white  precipitate  of  mercurous  sulphate. 

4.  The  reagent  is  made  by  dissolving  30  c.c.  of  mercury  in  570  c.c.  of 
concentrated  nitric  acid  and  diluting  with  twice  its  bulk  of  water.     It  contains 
mercurous  and  mercuric  nitrates,  excess  of  nitric  acid,  and  a  small  amount  of 
nitrous  acid. 

5.  The  reaction  should  never  be  attempted  with  a  strongly^alkaline  fluid, 
since  the  alkali  will  precipitate  the  yellow  or  black  oxides  of  mercury. 


4  THE     PROTEINS.  [CH.    I. 

6.  The   reaction   is   given   with   all   aromatic   substances  that    contain    a 
hydroxyl  group  attached  to  the  benzene  ring.     Thus  it  is  given  by  phenol, 
salicylic  acid,  and  naphthol,  but  is  not  given  by  benzoic  acid. 

7.  The  aromatic  substance  derived  from  protein  that  is  responsible  for  the 
reaction  is  tyrosine.          C^^^^-  NH2.COOH    (4) 

3.  The  glyoxylic  reaction  (Hopkins  and  Cole).  Treat  2 
or  3  c.c.  of  the  fluid  with  the  same  bulk  of  "reduced  oxalic  acid." 
Mix  and  add  an  equal  volume  of  concentrated  sulphuric  acid, 
pouring  it  down  the  side  of  the  tube.  A  purple  ring  forms  at  the 
junction  of  the  fluids.  Mix  the  fluids  by  shaking  the  tubes  gently 
from  side  to  side.  The  purple  colour  spreads  through  the  whole 
fluid. 

NOTES. — 1.     Reduced    oxalic  acid    is   prepared   by   one  of  the  following 
methods : — 

A.  Treat  half  a  litre  of  saturated   solution  of  oxalic   acid   with  40 
grammes  of  2  per  cent,  sodium  amalgam  in  a  tall  cylinder.     When  all  the 
hydrogen  has  been  evolved  the  solution  is  filtered  and  diluted  with  twice 
its  volume   of   distilled    water.       The   solution  now  contains  oxalic  acid, 
sodium  binoxalate,  and  glyoxylic  acid  (COOH.CHO).     It  should  be  kept 
in  a  closed  bottle  containing  a  little  chloroform. 

B.  In  a  flask  place  10  grammes  of  powdered  magnesium  and  just 
cover  with  distilled  water.     Slowly  add  250  c.c.  of  saturated  oxalic  acid, 
cooling  under  the  tap  at  intervals       Filter  off  the  insoluble  magnesium 
oxalate,  acidify  with  acetic  acid,  dilute  to  one  litre  with  distilled  water,'  and 
bottle  as  above. 

2.  The  reaction  does  not  succeed  in  the  presence  of  nitrates,  chlorates, 
nitrites,  or  excess  of  chlorides. 

3.  The   colour   is   not   well    seen   if   the   protein   is   mixed   with    certain 
carbohydrates  (eg.   cane-sugar),  owing  to   the   char  produced  by  the    strong 
sulphuric  acid. 

4.  It  is  most  important  to  use  pure  sulphuric  acid  for  this  test.     It  some 
times  fails  owing  to  the  presence  of  impurities  in  the  acid  employed. 

5.  In  performing  the  test  on  a  solid  substance,   like  fibrin  or  keratin,  a 
small   amount  of  the  material  should  be  heated  with  a  few  c.c.  of  the  reduced 
oxalic  acid  and  an  equal  volume  of  strong  sulphuric   acid.     The   mixture    is 
shaken,  and  as  the  protein  dissolves  in  the  strong  acid  both  the  fluid  and  the 
solid  particles  assume  a  purple  colour. 


CH.    I.] 


BIURET     REACTION. 


6.     The   substance   in    che   protein   molecule   that   is  responsible  for  the 
reaction  is  tryptophane  (indol-amino-propionic-acid)  Cn  Hi2  N2  C>2. 
CH 


HC 


NHa 
C.CH2.CH.COOH. 


CH 


NH 


4.  Piotrowski's  reaction  (the  biuret  reaction).  Treat 
5  c.c.  of  the  solution  with  an  excess  of  sodium  hydrate  and  a  drop 
of  a  1  per  cent,  solution  of  copper  sulphate.  A  violet  or  pink 
colour  is  produced. 

NOTES. — 1.  The  reaction  is  of  especial  importance  in  testing  fojr  albumoses 
and  peptones,  which  give  a  rose  colour.  It  is  generally  stated  that  other 
proteins  give  a  voilet,  but  usually  egg-albumin  gives  a  distinct  rose  tint*— 

2.  The  nomenclature  of  the  reaction  is  somewhat  varied.  Some  writers  use 
the  term  "  biuret  reaction"  even  when  performed  on  serum-proteins,  but  others 
restrict  this  term  to  the  reaction  given  by  albumoses  and  peptones,  employing 
the  term  "  Piotrowski's  reaction  "  to  that  given  by  albumins  and  globulins. 

3.  The   test   cannot   be   applied  in  the   presence   of   a  large  amount    of 
magnesium  sulphate,  owing  to  the  precipitation  of  magnesium  hydrate  by  the 
alkali. 

4.  If  the  solution  contains  much  ammonium  sulphate  it  muat  be  treated 
with  a  large  excess  of  strong  sodium  hydrate  as  directed  in  Ex.  54. 

5.  The  reaction  is  given  by  nearly  all  substances  containing  two  CONH 
groups  attached  to  one  another,   to  the  same  nitrogen  atom,   or  to  the  same 
carbon  acorn.     Thus  it  is  given  by 

CONH2        CONH2  CONH2 

CONH2  ~  S   NH        >        CH2    * 

|  I 

CONH2  CONH2 

Oxamide.  Biuret  (See  Ex.  253).          Malonamide. 

The  cause  of  the  reaction  with  proteins  is  the  presence  of  one  or  more  groupings 
of  the  last  type,  formed  by  the  condensation  of  the  carboxylic  group  of  an 
amino-acid  with  the  amino  group  of  another  amino-acid.  Thus  it  would  be 
given  by  the  polypeptide,  glycyl-alanyl-tyrosine 


CHs 

1 

C6H4.OH 

1 
CH2 

1 

NH2.CH2.CO. 

Glycyl- 

NH.CH.CO 
-alanyl- 

NH.CH.COOH 

-tyrosine. 

THE     PROTEINS. 


[CH.    I. 


5.  The  sulphur  reaction.     Boil  a  little  undiluted  egg-white 
or  serum  with  some  40  per  cent,  sodium  hydrate  for  two  minutes, 
and  then  add  a  drop  or  two  of  lead  acetate.    The  solution  turns  deep 
black. 

NOTES.— 1.  This  reaction  is  due  to  the  fact  that  the  sulphur  of  the  protein 
is  liberated  as  sodium  sulphide  when  boiled  with  the  strong  alkali.  The 
sulphide  gives  a  black  colour  or  precipitate  of  lead  sulphide  when  the  solution 
is  subsequently  treated  with  lead  acetate. 

2.  The  reaction  does  not  succeed  with  caseinogen,  peptones,  and  certain 
other  proteins. 

3.  The  sulphur  in  the  protein  is  mainly  combined  as 

CH2.SH  CH2.S.S.CH2 

I  I  I 

CH.NH2  or  CH.NH2  CH.NH2 

I  I  I 

COOH  COOH      COOH 

Cystein  Cystine 

6.  Molisch's  reaction.     Treat  5  c.c.  of  the  diluted  solution 
with    three   or  four  drops  of  a   1%  solution  of  alpha-naphthol  in 
alcohol.     Mix,  and  run  about  5  c.c.  of  concentrated  sulphuric  acid 
under  the  fluid.     A  violet  ring  is  formed  at  the  junction  of  the  two 
liquids. 

NOTES. — 1.  The  reaction  is  due  to  the  presence  of  a  carbohydrate  group 
(glucosamine)  in  the  protein.  This  is  converted  by  the  acid  to  furfurol,  which 
condenses  with  alpha-naphthol  to  give  the  purple  colour.  (See  notes  to  Ex.  76.) 

D.    The  Albumins  and   Globulins   of  Blood   Serum. 

Ox  blood  is  collected  into  pans  and  is  allowed  to  stand  till  clotting  is 
complete.  The  serum  that  excludes  is  pipetted  off  and  kept  in  the  ice  chest  till 
required. 

7.  Take  the  specific  gravity  by  floating  a  clean,  dry  urino- 
meter  in  a  cylinder  containing  the  serum,  and  noting  the  graduation 
where  the  stem  of  the  urinometer  is  level  with  the  surface  of  the 
fluid.     It  is  usually  about  1030  (water  being  taken  as  1000). 

8.  Take   the  reaction  of  the  serum  to  litmus    paper.     It    is 
alkaline. 

Heat-coagulation  of  albumins  and  globulins. 
When  a  solution  of  albumin  or  globulin  is  heated 
under    certain    conditions,   the    protein   separates   from 


CH.    I.]  HEAT     COAGULATION.  7 

solution    in    a    form    that   is   insoluble    in    water,   salt 
solutions,    dilute    acids    and   alkalies.      This    change   is 
known   as   "  heat-coagulation." 

It  seems  to  consist  of  two  processes : 

(1)  The  interaction  of  protein  and  water  ("  denatur- 

ation  "). 

(2)  The  subsequent  agglutination  and  separation  of 

the  product. 

The  first  process  may  take  place  without  the  second. 

Both  processes  are  much  affected  by  the  reaction  of 
the  solution  and  by  the  presence  of  neutral  salts. 

In  general  it  might  be  stated  that  an  increase  in 
acidity  or  alkalinity  up  to  a  certain  point  favours  de- 
naturation  but  decreases  the  tendency  to  agglutination. 
The  reverse  is  true  for  neutral  salts. 

The  best  medium  for  obtaining  heat-coagulation  is 
one  very  slightly  acid  and  containing  a  small  amount  of 
a  neutral  salt,  preferably  that  of  one  of  the  alkaline  earths, 
e.g.  calcium  chloride. 

The  material  produced  by  heating  the  protein  with 
water  can  be  regarded  as  a  hydrolytic  product,  meta- 
protein.  If  there  be  a  sufficient  amount  of  acid  or  alkali 
present  there  is  no  agglutination  of  this  unless  a  certain 
amount  of  neutral  salt  be  present.  In  general  it  can 
be  stated  that  the  smaller  the  amount  of  neutral  salt 
present,  the  smaller  is  the  amount  of  alkali  or  acid 
necessary  to  inhibit  agglutination. 

When  a  protein  is  treated  with  a  dilute  acid,  e.g.  HC1, 
a  salt  is  formed.  This  is  hydrolysed  by  water  into 
protein  and  free  HC1,  which  can  be  completely  removed 
by  prolonged  dialysis.  But  if  such  a  solution  of  protein  in 
weak  acid  be  boiled,  the  coagulum  that  forms  consists  of 
the  salt,  that  is,  the  HC1  is  partly  removed  from  the 
solution  on  coagulation. 


8  THE     PROTEINS.  |_CH.    I. 

As  regards  the  condition  of  the  protein  in  "  solution," 
it  has  been  shewn  that  the  particles  are  really  suspended 
in  the  "  solvent "  and  that  they  carry  an  electrical  charge. 
This  charge  determines  the  stability  of  the  system,  and 
any  factor  tending  to  reduce  the  charge  promotes  pre- 
cipitation or  coagulation.  The  sign  of  the  charge  on  the 
particle  is  determined  by  the  chemical  nature  of  the 
particle,  and  may  also  depend  on  the  nature  of  the  solvent. 
Hardy  has  shewn  that  in  the  case  of  the  proteins,  which 
have  amphoteric  characters,  the  sign  of  the  particle  is 
positive  when  the  fluid  is  acid  and  negative  when  the 
fluid  is  alkaline.  When  a  salt  is  added  to  such  a  colloidal 
solution  it  exerts  a  coagulative  effect  which  depends  upon 
one  of  its  ions,  th<3  coagulating^Jnn  being  that  which 
carries  a  charge  opposite  in  sign  to  that  of  the  particle. 
The  coagulative- power  increases  rapidly  with  the  valency. 
Thus  in  acid  solution  the  protein  has  a  positive  charge 
and  so  is  precipitated  by  negative  ions,  and  it  is  found 
that  the  potassium  salt  of  citric  acid  (trivalent)  is  much 
more  effective  than  the  potassium  salt  of  sulphuric  acid 
(divalent),  and  this  more  than  the  potassium  salt  of 
hydrochloric  acid.  In  alkaline  solutions  on  the  other 
hand  the  cation  is  the  coagulative  ion  and  cerium  chloride 
(Ce  Cla)  is  more  efficient  than  barium  chloride  (Ba  Cla)  and 
this  more  than  sodium  chloride  (Na  Cl). 

In  the  following  two  exercises,  the  explanations 
offered  in  the  notes  are  sufficient  for  elementary  students. 

9.     Dilute  5  c.c.  of  serum  with  45  c.c.  distilled  water. 

(a)  Boil  5  c.c.  in  a  test-tube.     The  solution  sometimes  be- 
comes opalescent,  but  no  definite  coagulum  is  formed.     Cool  the 
tube  and  add  1   per  cent,  acetic  acid  drop  by  drop.     A  precipitate 
of  metaprotein  is  formed,  soluble  in  excess  of  acid. 

(b)  Boil  5  c.c.  with  two  drops  of  1  per  cent,  acetic  acid.     A 
white  flocculent  precipitate  is  formed.     Cool  the  tube  ancUadd  two 


CH.    I.]  HEAT    COAGULATION.  9 

or  three   drops  of   strong    nitric    acid.     The    precipitate    does    not 
dissolve. 

(c)  Treat  5  c.c.  of  serum  with  -4  per  cent,  hydrochloric  acid, 
drop  by  drop,  till  the  solution  is  clear  (about  5  drops  are  usually 
necessary).     Boil.     A  precipitate  is  not   formed.     Cool    the    tube 
and  add  2  per  cent,  sodium  carbonate,  drop  by  drop.     A  precipitate 
is  formed,  which  redissolves  in  excess. 

(d)  Boil  5  c.c.  with  two  drops  of  2  per  cent.  Na2CO3.     A 
coagulum  is  not  formed.     Cool  the  tube  and  add  1  per  cent,  acetic 
acid.       A    precipitate  of  metaprotein  is  formed,  soluble  in  excess 
of    acid. 

NOTES. — 1.  These  reactions  are  of  very  great  importance,  and  are  to  be 
explained  as  follows :  Serum  contains  two  varieties  of  proteins,  known  as 
globulins  and  albumins,  which  are  coagulated  by  boiling,  provided  that  the 
reaction  of  the  fluid  is  neutral  or  very  faintly  acid.  If  the  solution  is  alkaline 
(and  it  must  be  remembered  that  serum  is  alkaline)  the  proteins  are  acted  on  by 
the  alkali  as  the  temperature  rises  and  are  converted  into  a  substance  known  as 
metaprotein,  which  is  not  coagulated  by  heat  when  in  solution.  If  the  reaction 
be  markedly  acid,  as  in  (c),  the  proteins  are,  similarly,  converted  to  metaprotein. 
But  if  the  reaction  be  neutral  or  only  faintly  acid,  as  in  (b),  a  coagulum  is  formed 
on  boiling.  This  coagulum  consists  of  the  whole  of  the  albumin  and  globulin, 
and  is  insoluble  in  water,  dilute  acids,  and  dilute  alkalies. 

2.  The  addition  of  the  nitric  acid  in  (6)  is  to  ensure  that  the  precipitate  that 
appears  on  boiling  does  not  consist  of  calcium  or  magnesium  phosphate,  which 
is  soluble  in  dilute  nitric  acid.  That  such  a  phosphatic  precipitate  can  be 
formed  on  boiling  certain  solutions  is  shown  by  the  following  experiment.  Treat 
a  solution  of  calcium  chloride  with  sodium  phosphate  and  then  with  excess  of 
sodium  carbonate.  A  precipitate  of  Ca3(PO4)2  appears.  Add  acetic  acid  drop 
by  drop  till  the  precipitate  just  dissolves  owing  to  the  formation  of  the  acid 
phosphate.  Boil  the  solution  for  half  a  minute.  A  white  precipitate  appears. 
Add  a  drop  or  two  of  nitric  acid.  The  precipitate  dissolves.  The  appearance 
of  the  precipitate  of  Ca3(PO4)2  on  boiling  is  due  to  the  alteration  of  reaction 
as  the  CO2  is  evolved. 

10.  Take  5  c.c.  of  the  diluted  serum  in  a  test-tube:  add  two 
drops  of  1  per  cent,  acetic  acid  and  place  the  test-tube  in  a  beaker 
of  water,  supporting  it  by  a  clamp  so  that  it  does  not  touch  the 
bottom  of  the  beaker.  Heat  the  water  with  a  Bunsen  flame  and 
note  the  temperature  at  which  coagulation  begins.  It  usually 
commences  at  about  70°  C.,  and  is  complete  at  82°  C.  It  takes 
place  chiefly  between  73°  and  75°  C. 


10  THE     PROTEINS.  [CH.    I. 

NOTE. — The  various  albumins  and  globulins  have  different  coagulating 
points,  but  since  this  point  varies  with  the  concentration  of  the  electrolytes  in 
the  solution  it  can  only  be  used  for  separating  and  distinguishing  proteins  when 
the  conditions  are  similar.  Nevertheless  the  coagulation  temperature  serves  to 
distinguish  myosin  (56°C.)  and  fibrinogen  (56°C.)  from  serum-globulin  (73°C.). 

11.  Place  about  4  c.c.  of  serum  in  a  test-tube  and  cool  to 
0°  C.  by  means  of  a  freezing  mixture.     Fill  the  tube  with  strong 
alcohol  that  has  previously  been  cooled  to  about  8°  C.,  and  mix.     A 
white   precipitate    of   the  proteins  is  formed.     Filter  at  once  and 
treat  the  precipitate  with  water.     It  dissolves. 

12.  Allow  a  few  drops  of  serum  to  fall  into  about   10  c.c.  of 
strong  alcohol  at  room  temperature.     A  white  precipitate  is  formed. 
Shake  well  and  allow  to  stand  for  half  an  hour.     Filter  and  treat 
the  precipitate  with  water.     It  does  not  dissolve. 

NOTE. — These  two  exercises  show  that  the  serum  proteins  are  first  pre- 
cipitated and  then  coagulated  by  strong  alcohol. 

13.  To   about    5    c.c.    of  diluted  serum  add  a  few  drops  of 
strong   acetic  acid.     A  precipitate  is  not  formed.     Now  add  four 
or  five  drops  of  strong  nitric  acid  :  a  white  precipitate  is  formed. 

NOTE. — The  serum-proteins  are  not  precipitated  by  acetic  acid  (thus 
differing  from  mucin,  caseinogen  and  nucleoprotein).  The  action  of  nitric  acid 
on  serum-proteins  is  to  produce  metaprotein,  which  is  insoluble  in  strong 
mineral  acids.  This  precipitability  of  albumins  and  globulins  is  the  basis  of 
Heller's  test  for  these  proteins  in  urine  (Ex.  278). 

Precipitation  by  Alkaloidal  Reagents. 

14.  Treat  5  c.c.  of  diluted  serum  with  two  or  three  drops  of 
strong   acetic  acid  and  two  drops  of  potassium  ferrocyanide.     A 
white   precipitate    is   formed.      Boil.      The    precipitate    does     not 
dissolve. 

NOTES. — 1.  Primary  albumoses  also  are  precipitated  by  potassium 
ferrocyanide  and  acetic  acid,  but  the  precipitate  produced  dissolves  on  warming 
and  reappears  on  cooling.  (See  Ex.  52.) 

2.  The  precipitate  and  fluid  often  become  coloured  blue-green  on  boiling. 
This  is  due  to  a  decomposition  of  the  hydro-ferrocyanic  acid  on  boiling  it  with 
certain  organic  substances,  such  as  proteins. 

15.  To    some    serum    diluted    about  ten  times  and   acidified 
with  dilute  hydrochloric  acid  add  a  few  drops  of  tannic  acid.     A 
white  or  brown  precipitate  is  formed. 


CH.    I.]  PROTEIN     PRECIPITANTS.  11 

16.  Treat  5  c.c.  of  diluted  serum  with  an  equal  volume  of 
Esbach's  solution.     A  yellowish  precipitate  is  formed. 

NOTE. — Esbach's  solution  is  prepared  by  dissolving  10  grms.  of  picric  acid 
and  10  grms.  of  citric  acid  in  water  and  making  the  solution  up  to  1  litre. 

17.  Acidify  some  diluted  serum  with  dilute  hydrochloric  acid 
and  add  a  few  drops  of  potassio-mercuric  iodide  (Brucke's  reagent). 
A  white  precipitate  is  formed. 

NOTE. — Brucke's  reagent  is  prepared  by  dissolving  50  grm.  of  potassium 
iodide  in  500  c.c.  water,  saturating  with  mercuric  iodide  (120  grm.),  and  making 
up  to  1  litre. 

18.  Acidify  5  c.c.  of  serum  with  dilute  hydrochloric  acid  and 
add   a    solution    of    phosphotungstic  acid.     A  white  precipitate  is 
produced. 

Precipitation  by  the  salts  of  the  Heavy  Metals. 

19.  To    diluted    serum  add  a  few  drops  of  mercuric  nitrate 
solution.     A    white    precipitate    is    formed,    soluble    in    saturated 
sodium    chloride    solution,    and    reprecipitated    from    this    by    the 
addition  of  dilute  hydrochloric  acid. 

20.  To  diluted  serum  add  ferric  chloride  solution.     A  precipi- 
tate is  formed  soluble  in  excess. 

21.  To  diluted  serum  add  copper  sulphate   solution  drop  by 
drop.     A  bluish-grey  precipitate  is  formed. 

22.  To  diluted  serum   add  a  solution  of  lead  acetate  or  basic 
lead  acetate.     A  white  precipitate  is  formed. 

The  remaining  exercises  of  this  section  deal  with  the  special 
physical  properties  of  the  globulins  and  albumins  of  serum. 

Globulins  are  insoluble  in  distilled  water,  but  soluble 
in  dilute  acids  and  alkalies,  and  in  weak  solutions  of 
neutral  salts. 

A  neutral  solution  in  a  dilute  salt  is  coagulated  on 
boiling. 

A  solution  in  dilute  acid  or  alkali  is  converted  into  a 
solution  of  metaprotein  on  boiling. 

If  the  globulin  be  dissolved  in  a  minimum  amount  of 
a  neutral  salt  solution  and  the  solution  be  diluted  with 


12  THE     PROTEINS.  [cH.    I. 

several  volumes  of  distilled  water,  the  globulin  is  partially 
precipitated,  for  a  certain  concentration  of  salt  is  necessary 
to  keep  the  globulin  in  solution.  If  the  globulin  be  dis- 
solved in  dilute  acid  or  alkali,  there  is  no  precipitation  on 
dilution. 

The  globulins  are  completely  precipitated  by  full 
saturation  with  magnesium  sulphate  or  by  half -satura- 
tion with  ammonium  sulphate,  i.e.  by  treatment  of  the 
solution  with  an  equal  volume  of  a  saturated  solution 
of  ammonium  sulphate. 

Albumins  are  soluble  in  distilled  water,  dilute  salt 
solutions,  dilute  acids  and  alkalies. 

A  neutral  solution  in  water  or  salt  is  coagulated  on 
boiling. 

A  solution  in  dilute  acid  or  alkali  is  converted  to  a 
solution  of  metaprotein  on  boiling. 

Solutions  of  albumins  are  only  partially  precipitated 
by  saturation  with  magnesium  sulphate  or  by  half- 
saturation  with  ammonium  sulphate  if  the  reaction  of 
the  solution  be  neutral  or  alkaline. 

They  are  more  completely  precipitated  by  solutions 
of  these  substances  in  the  presence  of  acid. 

They  are  completely  precipitated  by  full  saturation 
with  ammonium  sulphate  from  a  neutral,  acid,  or  alkaline 
solution. 

NOTE. — There  is  considerable  confusion  as  to  the  exact  differences  between 
globulins  and  albumins.  Some  writers  prefer  to  call  globulins  all  those  heat- 
coagulable  proteins  that  are  soluble  in  neutral  salts  and  are  precipitated  by  half- 
saturation  with  ammonium  sulphate.  Others  (including  the  author)  would 
limit  the  term  globulin  to  those  heat-coagulable  proteins  that  are  insoluble  in 
water  and  soluble  in  neutral  salt  solutions.  There  is  no  doubt  but  that  the 
precipitate  obtained  by  half  saturation  of  serum  with  ammonium  sulphate 
contains  at  least  two  substances,  one  insoluble  in  water  (precipitated  by 
dialysis),  the  other  soluble  in  water. 

Some  call  the  insoluble  body  "  eu-globulin  "  and  the  soluble  substance 
"  pseudo-globulin."  But  it  is  by  no  means  certain  that  this  latter  differs  at  all 


CH.    I.]  GLOBULINS.  13 

from  the  albumin  that  is  only  precipitated  by  full  saturation  with  ammonium 
sulphate.  Therefore  it  seems  better  at  present  to  restrict  the  term  globulin  to 
that  portion  of  the  serum  protein  that  is  insoluble  in  water. 

23.  Dilute  5  c.c.  of  serum  with  50  c.c.  of  distilled  water.     A 
faint  cloud  of  serum-globulin  is  formed.     Add  '4  p.c.  hydrochloric 
or  1   p.c.  acetic  acid,  drop   by   drop.     The  cloud  becomes  denser 
and  then  clears  up. 

NOTE. — The  globulin  in  the  serum  is  held  in  solution  both  by  salts  and 
dilute  alkalies.  Dilution  alone  produces  a  very  small  precipitate,  but  if  the 
solution  be  now  treated  with  just  sufficient  acid  to  neutralise  the  alkali,  a  much 
larger  fraction  of  the  globulin  is  thrown  down.  When  an  excess  of  acid  is 
added  the  globulin  dissolves. 

24.  Prepare    a    suspension    of    globulin    by    the   following 
method.     To    15   c.c.  of  serum  in  a  beaker  add  2  c.c.    (about  30 
drops)  of   1   p.c.  acetic  acid  and  100  c.c.  distilled  water.     Stir  and 
allow  the  mixture  to  stand  for  about  20  minutes.     A  precipitate  of 
globulin    settles   down.     Very    carefully   pour  off  the   supernatant 
fluid  and  divide  the  suspended  globulin  into  two  equal  portions  in 
clean  test-tubes.     With  these  perform  the  two  following  exercises. 

25.  Add  a  5  p.c.  solution  of  sodium  chloride,  drop  by  drop, 
till  the  globulin  has  just  dissolved.     Divide  the  solution  into  three 
portions,  A,  B  and  C. 

(a)  Boil.     The  protein  is  coagulated. 

(b)  Dilute   with    about    five    volumes   of    distilled  water. 
The  globulin  is  partially  reprecipitated. 

(c)  Treat  with  an  equal  volume  of  saturated  ammonium 
sulphate  solution.     The  globulin  is  reprecipitated. 

26.  Add  '4  p.c.  H  Cl,  drop  by  drop,  till  the  globulin  has  just 
dissolved.     Divide  the  solution  into  three  portions,  D,  E  and  F. 

(d)  Add  2  p.c.  sodium  carbonate  solution  till  the  globulin 
is    partially    reprecipitated    (one    or    two    drops    only    are 
necessary).     Now  add  a  few  drops  of  5  p.c.  sodium  chloride. 
The  precipitate  of  globulin  redissolves. 

(e)  Boil    the    solution.     The    protein    is   not    coagulated. 
Cool  under  the  tap  and  add  enough  2  p.c.  sodium  carbonate 


14  THE     PROTEINS.  [CH.    I. 

to  precipitate  the  metaprotein  that  has  been  formed  by 
boiling.  Now  add  a  few  drops  of  5  p.c.  sodium  chloride. 
The  precipitate  of  metaprotein  does  not  dissolve. 

(/)   Dilute    with    about    five    volumes    of    distilled   water. 
The  globulin  is  not  thrown  out  of  solution. 

27.  Mix  about    10  c.c.  of   undiluted  serum  with  an  exactly 
equal  quantity  of  a  saturated  solution  of  ammonium  sulphate.     A 
thick  white  precipitate  is  formed  consisting   of    the  whole  of  the 
globulin  and  a  portion  of  the  albumin.     Filter  through  a  dry  filter 
paper   into  a  dry   test-tube.      Label   the    filtrate    A.     Scrape    the 
precipitate    off  the  paper  and  treat   it  with  distilled  water.     The 
precipitate    dissolves,    the    ammonium    sulphate    adhering    to    it 
forming  a  dilute  salt  solution  which  allows  the  globulin  to  go  into 
solution.     Boil    a    portion    of    this    solution.     A   heat-coagulum  is 
formed. 

28.  Filtrate    A   contains   serum-albumin   in  the  presence   of 
half -saturated  ammonium  sulphate.    Apply  the  following  tests : 

(a)  Boil  a  portion.     A  heat-coagulum  is  formed. 

(b)  To   another  add  one   drop   of   strong   acetic   acid.     A 
white  precipitate  of  serum-albumin  is  formed. 

(c)  Grind  the  remainder  in  a  mortar  with  solid  (NH4)2SO< 
till  the  fluid  is  saturated.     A  white  precipitate  of  serum-albumin 
is   formed.     Filter    off  the    precipitate   and    test    the    filtrate    for 
proteins   either  by   boiling  or  by   the  glyoxylic    or   xanthoproteic 
reactions.     Proteins  are  absent,   showing  that  all  the   proteins  of 
serum  are  precipitated  by  complete  saturation  with  (NH4)2SO4. 

NOTE. — A  certain  test  for  albumin  in  a  solution  is  to  half-saturate  it  with 
ammonium  sulphate,  filter  off  any  precipitate  that  may  be  present  and  boil  the 
filtrate.  A  heat-coagulum  indicates  albumin. 

29.  Serum  has  been  dialysed  in  parchment  tubes  for  two  or 
three  days  against  repeated  changes  of  distilled  water.     Note  the 
heavy  precipitate  of  serum-globulin  that  has  fallen  to  the  bottom  of 
the  tube. 


CH.    1. 1  EGG-WHITE.  g  15 

30.  Dilute  5  c.c.  of  serum  with  five  times  its  volume  of  tap 
water,  add  a  drop  or  two  of  2  per  cent,  calcium  chloride  and  a  drop 
or  two  of  neutral  litmus.  Boil  the  mixture  in  a  porcelain  dish,  and 
whilst  boiling  cautiously  add  1  per  cent,  acetic  acid  till  the 
reaction  is  faintly  acid.  Filter,  and  test  the  filtrate  for  proteins  by 
the  usual  colour  tests.  If  the  operation  has  been  carried  out 
successfully  the  filtrate  will  be  found  to  be  nearly  free  from 
proteins. 

NOTE. — This  is  the  method  usually  employed  for  removing  albumins  and 
globulins  from  solutiou.  It  is  obvious  from  the  note  to  Ex.  9  that  a  certain 
amount  of  metaprotein  is  formed  when  the  fluid  is  first  boiled,  but  this  meta- 
protein  is  precipitated  by  the  acetic  acid,  and  the  precipitate  is  coagulated  at 
boiling  temperature  so  that  it  does  not  re-dissolve  in  the  very  slight  excess  of 
acid  that  is  subsequently  added.  (See  Chapter  I.,  H.) 

It  is  most  important  that  the  acid  should  be  added  slowly  and  not  in  any 
excess.  The  small  amount  of  calcium  chloride  added  aids  in  the  aggregation 
of  the  protein  on  boiling. 


E.     The   Chemistry   of  Egg-white. 

31.  In  egg-white  which  has  been  well  beaten  with  a  whisk 
(to  break  up  the  containing  membranes),  and  diluted  with  four  times 
its  volume  of  distilled  water,  note  a  precipitate  of  ovo-mucin  and 
globulin.  Perform  the  following  tests  : 

(a)     Take  the  reaction  to  litmus.     It  is  alkaline. 

(6)  Cautiously  neutralise  with  dilute  acetic  acid.  A  slight 
increase  in  the  precipitate  of  ovo-mucin  and  globulin  is  noticed. 
Remove  this  by  filtration  if  necessary,  and  with  the  filtrate  perform 
the  following  reactions  : 

(c)  Boil  a  portion.     A  coagulum  is  formed,  indicating  the 
presence  of  either  a  globulin  or  an  albumin. 

(d)  Make    another   portion    very    faintly    alkaline    by    the 
addition  of  a  drop  or  two   of  2  per  cent.  Na2CO8.     Now  add  an 
equal    bulk    of    saturated    (NH4)2SO4.     A    slight    precipitate    of 
globulin  or  albumin  is  formed.     Filter  this  off,  and  boil  a  portion 


16  THE     PROTEINS.  [cH.    I. 

of  the  filtrate  with  a  drop  of  1  per  cent,  acetic  acid.  A  coagulum 
of  albumin  is  formed.  Saturate  the  remainder  of  this  filtrate  with 
(NH4)2SO4  by  grinding  with  the  solid  in  a  mortar.  A  precipitate 
of  albumin  is  formed. 

(e)  Completely  remove  the  globulin  and  albumin  by  boiling. 
Filter  and  apply  Millon's  or  the  xanthoproteic  protein  test  to  the 
filtrate.  Protein  is  found  in  small  quantities.  This  protein  is 
known  as  ovo-mucoid.  It  is  not  coagulated  by  boiling,  or  precipi- 
tated by  acetic  acid.  It  is  precipitated  by  saturation  with 
(NH4)2SO4,  and  also  by  strong  alcohol. 

32.  The  crystallisation  of  egg-albumin.  (Hopkins' 
method.)  Separate  the  white  from  a  number  of  new-laid  eggs, 
taking  care  not  to  allow  any  of  the  yolk  to  mix  with  the  white. 
Measure  the  egg-white  and  churn  it  up  with  an  exactly  equal 
volume  of  a  neutral  fully-saturated  solution  of  ammonium  sulphate 
by  means  of  a  whisk,  adding  the  sulphate  in  portions  and  mixing 
thoroughly  after  every  addition.  Notice  the  strong  smell  of 
ammonia  that  is  evolved.  Filter  the  mixture  through  a  large 
pleated  filter-paper.  Measure  the  filtrate.  Take  100  c.c.  of  it  and 
cautiously  treat  it  with  10  per  cent,  acetic  acid  from  a  burette, 
noting  the  original  level  of  the  acid  in  the  burette.  Add  the 
acid  a  drop  or  two  at  a  time,  shaking  gently  the  whole  time, 
until  the  precipitate  produced  at  each  addition  no  longer  dis- 
solves on  shaking,  and  the  whole  mixture  is  rather  opalescent. 
This  point  is  usually  somewhat  difficult  to  determine,  owing  to  the 
large  number  of  air-bubbles  that  become  suspended  in  the  fluid  and 
closely  resemble  a  fine  precipitate.  When  you  are  satisfied  that  a 
permanent  precipitate  has  been  produced,  run  in  1  c.c.  of  the  acid 
in  addition  to  the  amount  already  added ;  a  heavy  white  precipitate 
is  thus  produced.  Note  the  amount  of  acid  that  has  been  used  for 
the  portion  of  100  c.c.,  and  treat  the  remainder  of  the  filtrate  with 
a  corresponding  amount  of  acid.  Mix  the  two  portions  thoroughly 
and  allow  to  stand  overnight.  Note  that  the  precipitate  has 
increased  somewhat  in  amount.  Mount  a  drop  of  the  suspension 


CH.    I.]  GLUCO-PROTEINS.  17 

on  a  slide,  cover  with  a  slip,  but  do  not  press.  Examine  under  the 
high  power  of  the  microscope,  and  note  the  aggregation  of  very  fine 
needles. 

The  albumin  can  be  recrystallised  by  filtering,  dissolving  in  as 
small  an  amount  of  water  as  possible,  filtering  again,  and  cautiously 
adding  to  the  filtrate  saturated  ammonium  sulphate  till  a  faint 
permanent  precipitate  is  produced.  If  the  mixture  be  allowed  to 
stand  for  some  hours  the  albumin  will  separate  out  as  fine  needles. 

NOTES. — 1.  For  the  experiment  to  succeed  it  is  absolutely  essential  that 
all  the  eggs  employed  be  perfectly  fresh.  One  rather  stale  egg  may  interfere 
with  the  crystallisation  of  a  large  number  of  fresh  eggs. 

2.  It  is  important  to  add  exactly  the  amount  of  acetic  acid  mentioned, 
that  is,  one  per  mille  above  the  amount  required  to  give  a  faint  permanent 
precipitate. 

3.  The  same  method  can  be  employed  for  the  crystallisation  of  serum- 
albumin  from  the  perfectly  fresh  serum  of  a  horse,  ass  or  mule. 


F.    The  Gluco-proteins. 

These  bodies  are  conjugated  proteins,  the  protein 
being  united  to  a  carbohydrate  group. 

They  consist  of  the  mucins  and  mucinoids  or  niucoids. 
The  mucins  are  found  in  connective  tissue  and  are 
secreted  by  certain  of  the  salivary  glands  and  various  parts 
of  the  alimentary  canal,  notably  the  large  intestine. 
Their  solutions  are  viscous.  They  are  soluble  in  dilute 
alkalies  and  are  precipitated  from  solution  by  acetic  acid 
the  precipitate  being  insoluble  in  excess  of  acetic  acid. 
They  are  also  soluble  in  0-1  per  cent,  hydrochloric  acid. 
On  hydrolysis  with  acids  the  sugar  group  is  split  off  and 
will  reduce  Fehling's  solution. 

The  mucoids  are  not  so  viscous  and  not  so  readily 
precipitated  by  acetic  acid,  the  precipitate  being  soluble 
in  excess.  They  are  found  in  ovarian  cysts  and  in  white 
of  egg  (See  Ex.  31  (e)). 

c 


18  THE     PROTEINS.  [CH.    I. 

Preparation  of  Mucin.  Mince  the  submaxillary  gland  of  an  ox,  grind 
with  sand  and  add  -1  per  cent.  NaOH  (1  litre  to  50  grams  of  the  moist 
gland).  Shake  well  in  a  large  bottle  from  time  to  time  and  leave  for  about 
half  an  hour.  Strain  through  muslin  and  filter  through  coarse  filter-paper. 
(This  crude  solution  should  not  be  prepared  too  long  before  use,  as  mucin 
loses  its  characteristic  properties  if  left  standing  with  alkalies.) 

33.  Add  acetic  acid  drop  by  drop.     A  stringy  precipitate  is 
formed,  insoluble  in  excess  of  the  acid. 

34.  Remove  the  precipitate  on  a  glass  rod,  wash  with  water, 
and  apply  the  usual  colour  reactions  for  proteins,  e.g.  xanthoproteic, 
glyoxylic,  and  Millon's.     They  are  all  given  by  mucin. 

35.  Treat  some  of  the  precipitate  with  -1  per  cent.  HC1.    The 
mucin  dissolves. 

36.  Treat  some  of  the  precipitate  with  2  per  cent.  Na2COs. 
The  mucin  dissolves. 

G.    The   Nucleoproteins   and   Nucleohistones. 

These  substances  are  conjugated  proteins,  the 
protein  being  in  combination  with  nuclein.  Nuclein  is 
a  protein  combined  with  nucleic  acid,  a  complex  body 
rich  in  phosphorus.  The  nucleoproteins  and  nucleo- 
histones  are  found  in  most  tissues  of  the  body,  notably 
in  those  rich  in  cells,  as  the  thymus,  lymphatic  glands, 
testes,  pancreas,  etc.  They  differ  in  the  nature  of  the 
protein  combined  with  nuclein.  In  the  nucleoproteins 
it  is  of  the  nature  of  a  peptone  :  in  nucleohistone  it  is  a 
histone.  (See  page  1.)  The  nucleic  acids  are  polyiiucleo- 
tides,  formed  by  the  condensation  of  a  certain  number 
of  nucleotides,  which  have  the  composition  of  a  simple 
nucleic  acid.  The  mononucleotides  consist  of  phosphoric 
acid  in  combination  with  a  micleoside,  a  compound 
formed  by  the  union  of  a  sugar  with  a  purine  or  a 
pyrimidine  group.  In  many  cases  the  sugar  is  a  pentose 
(d-ribose),  but  in  others  it  is  a  hexose  which  has  not  yet 
been  identified. 


CH.    I.]  NUCLEOPROTEINS.  19 

The  composition  of  the  nucleic  acid  obtained  from 
the  thymus  can  be  represented  as  follows  : 
Guanine  -  sugar  -  P205 

Thymine  -  Sugar  -  P205 

0 

I 
Cytosine  —  Sugar  —  P205 

Adenine  —  Sugar  —  P205 

Purine  Nucleoside 

Mononucleotide 

The    hydrolysis    of    nucleoproteins     is    effected    by 
gastric,  pancreatic  and  intestinal  juices,  and  by  certain 
ferments,  known  as  nucleases,  found  in  the  tissues.     The 
action  of  these  is  shewn  in  the  following  scheme  : — 
Nucleoprotein 
gastric  juice 


Nuclein  Protein 

pancreatic  juice 

Nucleic  Acid  Protein 

intestinal  and 
pancreatic  juices 

Nucleotides 

i 

intestinal  juice  and    '• 
nucleases 

~F  I 

Phosphoric  acid  Purine  nucleosides          Pyrimidine  nucleosides 

nucleases  nucleases 

I  _  I 

Purine  base        Sugar  Pyrimidine  base        Sugar 

The   purine   bases   found   are   guanine  and   adenine, 

which  are  converted  by  tissue  ferments  called  guanase 

and  adenase  to  xanthine  and  hypoxanthine  respectively. 


20 


THE     PROTEINS. 


[CH.    I. 


Hypoxanthine  can  be  oxidised  in  the  tissues  by  hypo- 
xanthine  oxidase  to  xanthine,  and  this  to  uric  acid  by 
xanthine  oxidase.  The  uric  acid  can  be  further  oxidised 
(especially  in  the  dog)  to  allantoin  by  the  ferment 
uricase. 

These  reactions  are  of  considerable  importance  in 
connexion  with  the  problem  of  the  origin  of  the  uric  acid 
excreted  by  the  mammal. 


N  =  C.NH2 

-  NH- 

N-  C-N 
adenine 


HN-CO 

H2N.6      C  -  NH 

ll       ii 
N-C-N 

guanine 


H2N 

OC    OC  -    NH 

L     1 
HN  -  CH  -  NH 

Allantoin 


adenase 


HN-CO 

HC      C - NH 

ll         II 
N  -  C  -  N 

hypoxanthine 


hypox  anthine-oxidase 


guanase 


HN-C-N 
xanthine 


xan 


thine-oxidase 


urcase 


HN-CO 
OC      C  -  NH- 


-  C  -  NH 
uric  acid 


Preparation.  Lymphatic  glands  of  the  ox  or  sheep,  or  the  thymus  of 
a  calf  are  freed  from  fat,  finely  minced,  ground  with  sand  and  extracted 
for  twelve  hours  with  ten  times  their  weight  of  distilled  water  in  a  large 
bottle,  a  small  amount  of  toluol  or  chloroform  being  added  to  prevent 
decomposition.  The  bottle  should  be  shaken  vigorously  at  frequent 
intervals  to  break  up  the  gelatinous  masses  that  sometimes  form.  The 
fluid  is  strained  and  centrifugalised  to  remove  all  debris  (nitration  being 
very  slow).  This  fluid  contains  both  nucleoprotein  and  nucleo-histone. 


CH.    I.]  NUCLEOPROTEINS.  21 

Physical  Properties.  Nucleoproteiiis  are  acidic  bodies 
which  dissolve  in  dilute  alkalies.  The  salt-like  bodies 
thus  formed  are  precipitated  as  the  free  acid  by  addition 
of  dilute  acetic  acid.  They  dissolve  to  an  opalescent 
solution  in  excess  of  strong  acetic  acid  (distinction  from 
mucin).  Nucleohistone  is  precipitated  as  a  calcium 
compound  by  -2  per  cent,  calcium  chloride  solution. 
Solutions  are  precipitated  by  half  saturation  with 
ammonium  sulphate. 

37.  To  a  portion  add  dilute  acetic  acid  till  no  more  precipitate 
is   produced,    and   place  on  the   water-bath   at    37°  C.    for  a   few 
minutes.     A  heavy  precipitate  of  nucleoprotein  and  nucleohistone 
is  formed.     Allow  this  to  settle  in  a  cylinder  :  pour  or  pipette  off 
as    much    of    the    supernatant    fluid    as    possible,    and    filter    the 
remainder.     Note  that  the  precipitate  is  soluble  in  dilute  alkalies 
and    is    reprecipitated    by    acidification ;    that    it    dissolves    to     an 
opalescent    solution    in    excess    of    acetic    acid    (difference    from 
mucin)  ;    and   that    it    gives    all    the    usual    colour    reactions    for 
proteins. 

38.  To  another  portion  add  one-tenth  of  its  volume  of  2  per 
cent,  calcium  chloride  and  warm  to  37°  C.     A  white  precipitate  of 
nucleohistone    is   formed.        Pour    off  the    supernatant   fluid,    and 
to    this    fluid    add    dilute    acetic    acid    drop    by    drop;    a    white 
precipitate  of  nucleoprotein  is  produced. 

39.  Precipitate    the    nucleoprotein    and    nucleohistone    from 
the  remainder  of   the  fluid  by  means  of  acetic  acid  as  in  Ex.  37. 
Collect  the  precipitate  on  a  filter  paper,  allow  it  to  drain  well,  and 
then  transfer  it  by  means  of  a  spatula  to  a  small  thimble-shaped 
porcelain  capsule.     Heat  carefully,  first  to  drive  off  the  water,  and 
then  to  carbonise  the  residue.      Add  one-third  of  a  crucible  full  of 
fusion    mixture  (K2CO3  two  parts,   KNO3  one  part),  and  heat  as 
strongly  as  possible  till  the  mass  fuses.     Allow  the  melt  to  cool, 
and    extract    it    with  nitric  acid    (diluted   with   an   equal  quantity 
of  distilled  water)  till  the   mixture  no  longer  effervesces.     Filter : 


22  THE     PROTEINS.  I.CH.    I. 

treat  the  filtrate  with'  about  one-tenth  its  volume  of  strong  nitric 
acid  and  one-third  its  volume  of  ammonium  molybdate  :  boil  for 
two  minutes.  The  yellow  crystalline  precipitate  separating  out 
on  the  sides  of  the  tube  shows  that  nucleoproteins  and  nucleo- 
histone  contain  phosphorus,  that  has  been  oxidised  to  a  phosphate 
by  the  fusing. 

H.    The  Metaproteins. 

The  metaproteins  are  derived  from  the  albumins 
and  globulins  by  hydrolysis.  This  can  he  effected 
rapidly  by  dilute  acids  and  alkalies  at  temperatures 
over  60°  C.  (see  notes  to  Ex.  9) :  more  slowly  at  body 
temperature.  They  are  formed  immediately  by  the 
action  of  strong  mineral  acids  at  room  temperature. 
(See  Exs.  1  and  18.)  They  are  insoluble  in  water,  strong 
mineral  acids,  and  all  solutions  of  neutral  salts,  but  are 
soluble  in  dilute  acids  or  alkalies  in  the  absence  of  any 
large  amount  of  neutral  salts.  They  are  not  thrown  out 
of  solution  (in  acid  or  alkali)  by  boiling.  But  if  such 
a  solution  be  neutralised  or  precipitated  by  the  addition 
of  an  excess  of  a  neutral  salt,  the  suspended  metaprotein 
is  coagulated  on  boiling,  so  that  it  will  no  longer  dissolve 
in  acid  or  alkali. 

Preparation.  Egg  white  or  serum  is  diluted  with  ten  times  its 
volume  of  either  -4  per  cent,  hydrochloric  acid  or  -1  per  cent,  sodium 
hydrate  and  the  mixture  placed  in  a  water  bath  01  incubator  at  40V  C.  for 
about  twenty-four  hours.  The  albumins  and  globulins  are  hydrolysed  to 
metaprotein. 

40.  To  about  twenty-five  c.c.  add  a  few  drops  of  litmus  and 
carefully  neutralise  with  2  p.c.  Na2CO8  or  4  p.c.  HC1.  A  bulky 
precipitate  of  metaprotein  separates  out.  Filter.  Scrape  the 
precipitate  off  the  paper  and  suspend  it  in  a  test-tube  about  half-full 
of  water.  Divide  the  suspension  into  six  equal  portions  and  with 
them  perform  the  following  six  exercises  : 


CH.    I.]  ALBUMOSES    AND    PEPTONES.  23 

41.  Add    some    *4    p.c.    HC1.      The    precipitate    dissolves. 
Neutralise  with  Na2CO:$ :  the  precipitate  reappears. 

42.  Add   concentrated  HC1  drop  by  drop.     The    precipitate 
dissolves  with  the  first  drop,  and  reappears  when  an  excess  is  added. 

43.  Dissolve    in    a    little    -4    p.c.    HC1.     Boil    the    solution : 
a  coagulum  is  not  formed.     Cool  under  the  tap  and  neutralise  with 
'2    p.c.   Na2CO3.     A   precipitate  is  formed  which  is  soluble  in  an 
excess. 

44.  Boil.     Cool  and  add  some  -4  p.c.  HC1.     The  precipitate 
does  not  dissolve,   i.e.  metaprotein    is    coagulated  when   boiled  in 
suspension. 

45.  Add  a  saturated  solution  of  ammonium  sulphate  drop  by 
drop.     The  precipitate  does  not  dissolve  in  any  dilution  of  the  salt. 

46.  Dissolve  in  a  little  -4  p.c.  HC1.     Treat  the  solution  with 
an  equal  volume  of  saturated  ammonium  sulphate  solution.     The 
protein  is  precipitated. 

I.     The  Albumoses  and  Peptones. 

These  hydrolysed  proteins  are  obtained  by  the  further 
action  of  acids  or  alkalies  on  globulins,  albumins  and 
nietaproteins.  They  are  best  formed  by  the  action  of 
pepsin  and  hydrochloric  acid  on  these  proteins.  Peptone 
is  the  end  product  of  gastric  digestion. 

They  are  prepared  on  a  commercial  scale  and  sold  as — 

(i.)  Witte's  peptone,  which  is  prepared  from  fibrin 
and  consists  of  a  mixture  of  albumoses  and 
peptone. 

(ii.)  Savory  and  Moore's  peptone,  which  is  prepared 
from  meat,  and  only  contains  traces  of 
albumoses. 

The  following  scheme  indicates  the  successive  steps 


24 


THE     PROTEINS. 


[CH.    I. 


in   the  digestion   of  fibrin  by  pepsin   and  0*2   per  cent, 
hydrochloric  acid : — 

Fibrin 

Soluble  Globulin 
Metaprotein 


Primary  albumoses  Secondary  albumoses 

Proto-albumose  :  Hetero-albumose.        Thio-albumose :  Synalbumose,  etc. 


Peptones. 

The  following  scheme  shews  the  method  adopted  for 
the  isolation  of  certain  of  the  albumoses  : — 

Neutral  Witte's  peptone,  treated  with  equal  volume  of 
saturated  ammonium  sulphate  solution. 


Precipitate :  dis- 
solved in  water. 
Treated  with  2 
volumes  of  strong 
alcohol. 


Ppt. 

Hetero- 
albumose. 


Filtrate. 

Proto- 
albumose 


Filtrate,  treated  with  half  its  volume  of  saturated 
ammonium  sulphate. 


Ppt.  dissolved 

in  water. 
Treated  with 

2  volumes  of 

alcohol. 

Ppt. 

Thio- 
albumose. 


Filtrate.     Saturated  with  ammo- 
nium sulphate. 


Ppt.  dissolved  in  water. 

Treated  with  2  volumes 

of  strong  alcohol. 


Filtrate. 


Peptones. 


Ppt. 
Neglect. 


Filtrate. 

Treated  with 
|  vols.  of  alco- 
hol. 


Ppt. 
Synalbumose. 


The  primary  albumoses  are  soluble  in  water,  dilute 
acids,  alkalies  and  salt  solutions.  Their  solutions  are  not 
coagulated  on  heating.  They  are  precipitated  by  half- 


CH.    I.]  ALBUMOSES    AND    PEPTONES.  25 

saturation  with  ammonium  sulphate.  They  give  a  pre- 
cipitate, that  disappears  on  warming  and  reappears  on 
cooling,  either  with  nitric  acid  or  potassium  ferrocyanide 
and  acetic  acid.  They  also  give  a  precipitate  in  the  cold 
with  copper  sulphate. 

They  give  all  the  ordinary  protein  colour  reactions, 
with  the  exception  of  Molisch's. 

The  secondary  albumoses  have  somewhat  similar 
properties  to  those  of  the  primary  albumoses :  hut  they 
are  not  precipitated  by  nitric  acid,  hydro-ferrocyanic  acid, 
or  copper  sulphate. 

They  require  more  than  half -saturation  with  ammo- 
nium sulphate  to  precipitate  them,  but  are  completely 
precipitated  by  full  saturation.  Thio-albumose  gives  all 
the  protein  colour  reactions  and  is  particularly  rich  in 
sulphur  (hence  its  name). 

Synalbumose  gives  the  protein  reactions,  with  the 
exception  of  the  glyoxylic  test. 

The  peptones  are  very  soluble  proteins  of  rather  a  low 
molecular  weight,  so  that  they  slowly  diffuse  through 
parchment  membranes.  They  are  the  only  proteins  not 
precipitated  by  full  saturation  with  ammonium  sulphate. 
They  fail  to  give  precipitates  with  Esbach's  or  Briicke's 
reagents  or  hydro-ferrocyanic  acid,  but  are  precipitated 
by  other  protein  precipitants,  as  tannic  acid,  phospho- 
tungstic  acid  and  lead  acetate. 

For  the  following  reactions  make  a  5  per  cent,  solution  of  "Witte's 
peptone"  in  hot  water,  just  acidify  with  acetic  acid  and  filter  from  a 
small  amount  of  insoluble  material  (nuclein?).  The  solution  contains  all 
the  albumoses  and  peptones. 

47.  Dilute  a  small  amount  with  three  or  four  times  its  bulk 
of  water,  and  to  portions  of  this  apply  the  usual  colour  reactions  for 
protein.  They  are  all  obtained.  Note,  in  particular,  that  the  biuret 
test  gives  a  rose  colour. 


26  THE     PROTEINS.  [cH.    I. 

48.  Boil  the  solution  with  a  trace  of  acetic  acid  :  it  does  not 
form  a  coagulum. 

49.  Add  a  little  tannic  acid  :  a  white  precipitate  is  formed. 

50.  Add  a  little  Esbach's  or  Briicke's  solution  :  a  yellow  or 
white  precipitate  is  formed. 

51.  Add  a  little  lead  acetate  solution:  a  white  precipitate  is 
formed. 

52.  To  10  c.c.  of  the  5  per  cent,  solution  in  a  small  beaker 
add    10  c.c.   of  a  saturated   solution    of    ammonium    sulphate.     A 
white    precipitate    of    the  primary  albumoses  is  formed.     Stir  the 
mixture  vigorously  for  a  short  time  with  a  glass  rod  that  has  one 
end  covered  with  a  small  piece  of  rubber  tubing  :  allow  to  stand  for 
a  few  minutes.     The  precipitate  will  usually  gather  together  and 
can  be  almost  completely  collected  as  a  gummy  mass  on  the  end  of 
the  rod.     Transfer  it  to  about  5  c.c.  of  hot  water.     The  precipitate 
dissolves.     Cool  the  solution  and  divide  it  into  three  portions. 

(a)  Add  a  drop  of  strong  acetic  acid  and  two  drops  of  potassium 
ferrocyanide.     A  white  precipitate  is  formed,  which  disappears  on 
heating  and  reappears  on  cooling. 

(b)  To  another  portion  add  a  few  drops  of  strong  nitric  acid. 
A   white   precipitate  is   formed,   which   disappears  on  heating  and 
reappears  on  cooling. 

(c)  To  the  third  portion  add  a  drop  of  copper  sulphate  solution. 
A  white  precipitate  is  formed. 

53.  The  fluid  from  which  the  main  mass  of  primary  albumoses 
has  been  removed  is  filtered  and  treated  in  a  beaker  with  a  single 
drop  of  sulphuric  acid,  and  then  with  ammonium  sulphate  that  has 
been  finely  powdered  in  a  mortar.     The  mixture  is  stirred  vigorously 
till  the  fluid  is  saturated  with  the  salt.     A  flocculent  precipitate  of 
the    secondary  albumoses  (deutero-albumoses)  is  formed.     Collect 
this   on  the  rod  as   before,  dissolve  in   a  little    water,   divide   the 


CH.    I.]  PEPTONES.  27 

solution  into  three  parts,  and  repeat  the  three  tests  already  per- 
formed with  the  primary  albumoses.  A  precipitate  is  not  formed  by 
any  of  the  reagents. 

54.  The  fluid  from  which  the  secondary  albumoses  have  been 
removed    contains    peptone.     Filter  it,  and  treat  a  portion  of  the 
filtrate  with  twice  its  volume  of  40  per  cent,  sodium  hydroxide  and  a 
drop  of  1  per  cent,  copper  sulphate.     A  pink  colour  appears,  due  to 
the  presence  of  peptone. 

Important  Note.  — This  large  excess  of  strong  NaOH  must  be  added  in 
order  to  decompose  the  (NH4)2SO4  with  which  the  solution  is  saturated.  The 
characteristic  rose  colour  is  only  obtained  when  the  alkalinity  is  due  to  NaOH, 
ammonia  being  quite  inefficient. 

5  c.c.  of  saturated  (NH4)2SO4  solution  contains  about  3'75  grms.  of  the  salt. 
This  requires  2  27  grms.  of  NaOH.  10  c.c.  of  40%  NaOH,  containing  4  grms. 
of  NaOH,  is  thus  sufficient. 

55.  Evaporate  a  small  portion  of  the  original  fluid  to  complete 
dryness,  finishing  the  process  on  a  water  bath  in  order  to  prevent 
charring.     Rub  up  the  residue  with  successive  small  quantities  of 
strong  alcohol  (95  per  cent.).     Add  the  extracts  together,  filter  and 
evaporate  them  to  dryness  on  a  water  bath.     Dissolve  the  residue 
from  this  evaporation  in  a  little  water  and  test  for  proteins  by  the 
various  colour  tests.     Only  insignificant  traces  are  present,  showing 
that  albumoses  and  peptones  are  insoluble  in  strong  alcohol. 

NOTE. — It  is  frequently  desirable  to  remove  all  proteins  from  a  solution 
before  testing  for  certain  substances,  e.g.  sugars,  bile-salts,  urea,  etc.  In  the 
case  of  albumoses  and  peptones  this  can  only  be  effected  by  the  method 
described  above,  advantage  being  taken  of  the  solubility  of  sugars,  etc.,  in 
alcohol,  and  the  insolubility  of  all  proteins  in  the  same.  The  aqueous  solution 
prepared  in  this  way  will  be  spoken  of  as  "  an  alcoholic  extract." 

Peptones.     Use  a  2  per  cent,   solution  of  Savory  and 
Moore's  peptone,  which  is  usually  free  from  albumoses. 

56.  Apply  the  usual  colour  reactions  for  proteins.     They  are 
all  obtained. 

NOTE.— The  glyoxylic  reaction  may  not  be  very  intense,  owing  to  the 
presence  of  chlorides  in  the  preparation.  Pure  peptone,  when  freed  from 
chloride  by  appropriate  means,  gives  a  very  good  glyoxylic  reaction. 


28  THE     PROTEINS.  [cH.    I. 

57.  Add  a  drop  or  two  of  strong  acetic  acid  and  a  drop  of 
potassium  ferrocyanide.     No  precipitate  is  produced,  showing  that 
the  primary  albumoses  are  absent. 

58.  Add   a   little    Esbach's    or    Brucke's    solution.     A    very 
slight    or  no   precipitate  is   formed,  if  the  solution   be    free    from 
albumoses. 

59.  Saturate   a   portion   with    (NH4)2SO4.       No    precipitate 
is  produced,  showing  that  all  albumoses  are  absent. 

60.  Treat  5  c.c.  of  the  nitrate  from  Ex.  59  with  two  volumes 
of  40  per  cent.  NaOH   and  a  drop  of  copper  sulphate.     A  pink 
colour  is  obtained. 

61.  Add  a  few  drops  of  a  solution  of  tannic  acid.     A  white 
precipitate  is  formed. 

62.  Add  a  few  drops  of  a  solution  of  lead  acetate.     A  white 
precipitate  is  formed. 

J.  %  The  reactions  of  certain  Sclero-proteins. 

Gelatin  is  found  in  the  body  in  the  form  of  its 
anhydride,  collagen.  This  occurs  in  white  fibrous  tissue 
and  in  the  organic  substance  of  bones,  and  can  be 
converted  into  gelatin  by  boiling  with  a  dilute  acid.  Dried 
gelatin  swells  in  cold  water,  but  is  quite  insoluble  in  it. 
On  warming,  a  more  or  less  viscid  solution  is  obtained, 
which  solidifies  to  a  jelly  on  cooling  provided  the  con- 
centration be  greater  than  1  per  cent.  This  process  is 
reversible  on  warming  and  cooling.  It  is  precipitated  be- 
half-saturation with  ammonium  sulphate,  by  tannic 
acid,  phospho  -  tungstic  acid,  Esbach's  and  Brucke's 
reagents,  but  not  by  normal  lead  acetate.  On  complete 
hydrolysis  it  yields  a  high  percentage  of  its  nitrogen  in  the 
form  of  glycine,  but  very  little  in  the  form  of  the  aromatic 
amino-acids,  tyrosine  or  tryptophane,  and  none  as  the 


CH.    I.]  GELATIN.  29 

sulphur-containing  compound,  cystine.  Therefore  its 
sohitions  fail  to  give  the  glyoxylic,  Millon's  and  sulphur 
colour  tests  for  proteins,  and  only  give  a  slight  xantho- 
proteic  test,  which  is  due,  either  to  an  impurity  or  to  a 
small  amount  of  phenyl-alanine. 

63.  Break  gelatin  up  into  small  pieces  and  add  a  small 
amount  of  cold  water.  The  gelatin  does  not  dissolve.  Immerse 
the  test  tube  in  a  beaker  of  boiling  water  and  leave  it  for  a  short 
time.  The  gelatin  dissolves.  Cool  the  tube  under  the  tap  :  the 
gelatin  sets  to  a  jelly.  Perform  the  following  tests  with  an 
approximately  1  per  cent,  solution  of  gelatin : 

(a)  Xanthoproteic  reaction  :  slight. 

(6)  Millon's  reaction :  very  slight,  showing  absence  of  tyrosine 
from  gelatin  molecule.  (See  Notes  to  Ex.  2.) 

(c)  Glyoxylic    reaction:    not    obtained,    showing    absence    of 
tryptophane.     (Ex.  3.) 

(d)  Biuret  reaction  :  violet  colour. 

(e)  Sulphur  reaction  :  not  obtained,  showing  absence  of  cystine. 

(Ex.  5.) 

(/)  Add  acetic  acid  :  no  precipitate. 

(g)  Add  acetic  acid  and  potassium  ferrocyanide  :  very  slight  or 
no  precipitate. 

(h)  Add  tannic  acid :  white  precipitate. 

(i)  Add  lead  acetate :  very  slight  or  no  precipitate. 

(/)  Half  saturate  with  ammonium  sulphate.  The  whole  of 
the  gelatin  is  precipitated,  as  shown  by  a  negative  biuret  test  in  the 
filtrate  (distinction  from  peptones). 

(k)  Add  Esbach's  or  Briicke's  solution :  yellow  or  white 
precipitate  (distinction  from  peptones). 


30  THE     PROTEINS.  [CH.    1. 

Keratin.  An  insoluble  body  found  in  the  hair,  skin, 
nails,  and  horns.  Remarkable  for  the  high  percentage  of 
cystine  it  yields  on  acid  hydrolysis. 

60.  Perform  the  following  tests  by  using  horn  shavings, 
or  hair.  Note  insolubility  in  hot  or  cold  water,  dilute  acids,  and 
dilute  alkalies. 

(a)  Xanthoproteic  reaction  :  well  marked. 

(b)  Millon's  reaction :  well  marked. 

(c)  Glyoxylic  reaction  :  well  marked. 

'(d)  Biuret  reaction :  not  obtained,  owing  to  insolubility. 
(e)  Sulphur  reaction  :  well  marked. 


CHAPTER  II. 
THE    CARBOHYDRATES. 

These  compounds  contain  the  elements  carbon, 
hydrogen  and  oxygen,  the  general  formula  being 
Cx(HaO)y.  They  can  be  sub-divided  into  several  groups. 

A.  The  Monosaccharides. 

B.  The  Disaccharides. 

C.  The  Polysaccharides. 

A.     The  Monosaccharides. 

The  monosaccharides  are  the  simplest  carbohydrates, 
and  all  the  others  can  be  hydrolysed  to  two  or  more 
molecules  of  monosaccharide  by  means  of  acids  or  certain 
ferments. 

They  consist  of  primary  alcoholic  (  — CH2OH)  or 
secondary  alcoholic  (  =  CH.OH)  groups  linked  to  an 
aldehyde  (-CHO)  or  ketone  (  =  C  =  0)  group.  Those  with 
an  aldehyde  group  are  called  aldoses  ;  those  with  a  ketone 
group,  ketoses.  They  contain  from  two  to  nine  carbon 
atoms  and  are  called  bioses,  trioses,  tetroses,  pentoses, 
hexoses,  etc.,  depending  on  the  number  of  carbon  atoms  in 
the  molecule. 

The  lower  members  of  the  series  are  not  important 
physiologically.  The  pentoses  C5H10O5  are  found  in  the 
urine  in  certain  pathological  conditions.  They  form  a 
constituent  part  of  the  molecule  of  nucleic  acid.  (See 
page  18.)  The  most  important  pentoses  are  the  aldoses 
arabinose  and  xylose,  obtained  from  gum  arabic  and 
pine-wood  or  straw  respectively  and  ribose,  obtained  by 
the  hydrolysis  of  the  nucleoproteins. 


32 


THE     CARBOHYDRATES. 


TCH.  ii. 


The  hexoses,  C6H]2O6,  are  of  great  physiological  import- 
ance. Of  the  many  that  have  been  synthesised  in  the 
laboratory  only  the  following  are  found  in  nature  and 
are  of  physiological  interest : — 


CHO 

CHO 

CHO 

CH2OH 

H.C.OH 

HO.C.H 

1 
H.C.OH 

CO 

HO.C.H 

HO.C.H 

HO.C.H 

H0.6.H 

1 

H.C.OH 

H.C.OH 

HO.C.H 

H.C.OH 

H.C.OH 

H.C.OH 

H.C.OH 

H.C.OH 

H2OH 

d- glucose  : 
(dextrose,  grape- 
sugar) 


CH2OH 

d-mannose 


CH2OH 

d-galactose 


CH2OH 

d-fructose 
(laevulose,  fruit 
sugar) 


It  will  be  noticed  that  the  first  three  are  aldoses, 
whilst  fructose  is  a  ketose. 

* 

The  first  three  are  stereo-isomers,  differing  only  in  the 
arrangement  of  the  H  and  OH  groups  in  space  round  the 
four  central  carbon  atoms,  all  of  which  are  asymmetric. 
(See  page  79.)  It  therefore  follows  that  these  compounds 
are  optically  active,  that  is,  their  solutions  can  rotate  the 
plane  of  polarised  light. 

In  the  above  formula  they  are  represented  as  being 
aldehydes,  but  certain  facts  seem  to  indicate  that  they  can 
exist  in  another  form.  Thusrtf  glucose  be  dissolved  in 
water  it  is  found  that  the  solution  at  first  has  a  much 
higher  rotatory  power  than  when  it  has  been  kept  for 
some  hours  or  has  been  boiled  with  a  trace  of  alkali. 
This  phenomenon  is  known  as  mutarotation.  Also  it  is 
very  much  less  active  chemically  than  the  above  formula 
warrants. 


CH.    II.] 


GLUCOSE. 


33 


These  properties  are  explained  by  assuming  that  when 
first  dissolved  in  water,  glucose  exists  as  a  y-lactoije, 
having  the  formula 

H-C.OH 
H.C.OH 
HO.C.H 


,OH 


In  this  state  the  *C  atom  is  asymmetric,  so  that  two 
forms  of  glucose  are  possible,  called  a-  and  /3-glucose. 

Under  certain  conditions  two  forms  of  glucose  can  be 
isolated,  one  with  a  rotary  power  of  110°,  the  other  with  a 
rotation  of  19°.  When  kept  in  solution  both  finally  attain 
a  rotation  of  52-5°. 


H.C.OH 


CH9OH 


JH2OH 
a-glucose.  ^-glucose. 

In  solution  both  forms  slowly  pass  into  the  aldelhyde 
form  (tautomerism).  If  the  *H  atom  be  replaced  by  some 
other  group  (generally  aromatic),  the  compound  formed 
is  called  an  a-  or  /3-glucoside,  which  can  be  converted 
into  glucose  and  another  compound  by  hydrolysis  with 
acids  or  certain  ferments. 


34  THE     CARBOHYDRATES.  [CH.    II. 

The  natural  glucosides  (phloridzin,  salicin,  etc.)  are 
/3-glucosides. 

Physical  properties  of  the  monosaccharides.  They  are 
white  crystalline  solids,  very  soluble  in  water  and  alcohol. 
Insoluble  in  ether,  acetone  and  most  of  the  organic 
solvents. 

They  are  optically  active,  the  natural  sugars  having 
the  following  rotatory  powers — 

Glucose  -   +  52-5°.          Galactose  =    +  82°. 

Fructose  -   93*8°. 

Chemical  properties.  Being  adehydes  or  ketones,  they 
are  susceptible  of  being  oxidised  to  various  acids,  thus 
reducing  certain  oxidising  reagents.  This  reaction  only 
takes  place  in  hot  alkaline  solutions,  and  is  of  great  value 
as  a  test  for  these  sugars,  and  especially  as  a  basis  of 
various  methods  of  estimation. 

They  react  with  phenyl  hydrazine  in  excess  to  give 
insoluble  crystalline  bodies  called  osazones.  These  are 
of  the  greatest  value  in  determining  the  presence  of  and 
in  characterising  the  monosaccharides,  though  not  in 
distinguishing  them  from  one  another. 

When  heated  with  an  alkali  the  monosaccharides 
become  yellow  and  then  brown,  and  finally  decompose 
into  a  mixture  of  acids  and  resinous  substances. 

They  are  reduced  by  sodium  amalgam  to  hexahydric 
alcohols.  Sorbite  is  formed  from  glucose,  mannite  from 
mannose  and  dulcite  from  galactose.  Fructose  yields  a 
mixture  of  sorbite  and  mannite. 

On  oxidation  glucose  gives  rise  to  three  acids— 
CO2H  CHO  CO2H 

(CHOH)4  (CHOH)4  (CHOH)4 

d)H2OH  C02H  C02H 

Gluconic  acid.     Glycuronic  acid.     Saccharic  acid. 


CH.-II.J  GLUCOSE.  35 

Glycuronic  acid  is  interesting  physiologically,  as  it  is 
frequently  found  in  the  urine  in  combination  with  various 
drugs,  such  as  chloral,  camphor,  phenol,  etc.,  in  the  form 
of  a  glucoside.  These  compounds  protect  the  organism 
from  the  injurious  effects  of  the  drugs. 

Glucose  (dextrose  or  grape-sugar).  Use  a  -2  per  cent, 
solution  for  the  following  reactions. 

65.  Boil  with  a  little  sodium  hydroxide.     The  solution  turns 
yellow.     (Moore's  test.) 

NOTE. — The  yellow  colour  is  due  to  the  formation  of  caramel  (a  condensa- 
tion product)  by  the  hot  alkali.  It  must  be  noted  here  that  glucose  is  com- 
pletely destroyed  by  prolonged  boiling  with  alkali. 

66.  Treat  two  or  three  c.c.  of  5  per  cent,  caustic  soda  with 
four  or  five  drops  of  a  1  per  cent,  solution  of  copper  sulphate.     A 
blue  precipitate  of  cupric  hydroxide,  Cu(OH)2,  is  formed.     Add  to 
the  mixture  an  equal  bulk  of  the  sugar  solution.     The  precipitate 
dissolves.     Boil  the  solution   for  a  short   time.     The   blue  colour 
disappears,  and  is  replaced  by  a  yellow  or  red  precipitate  of  cuprous 
oxide,  Cu2O.     (Trommer's  test.) 

NOTES — 1.  The  amount  of  copper  necessary  depends  on  the  percentage  of 
sugar  present.  If  only  a  small  amount  of  sugar  be  present  a  mere  disappearance 
of  the  blue  colour  is  all  that  may  happen,  or  possibly  the  fluid  may  assume  a 
faint  yellowish-red  tint.  If  excess  of  copper  be  added,  the  reduction  is  obscured 
by  the  blue  cupric  hydrate  in  solution,  or  the  black  precipitate  of  cupric  oxide 
that  is  formed  on  heating  this  in  the  alkaline  solution.  It  is  always  best  to  add 
the  copper  sulphate  a  few  drops  at  a  time,  boiling  between  each  addition. 

2.  The   reaction   is  a  type  of  several  that  have  been  introduced  for  the 
detection  of  glucose,  all  of  which  depend  on  the  fact  that  in  alkaline  solution  it 
has  reducing  properties  when  boiled.     For  this  reason,  glucose,  and  all  sugars 
that  have  this  property  are  sometimes  spoken  of  as  "  reducing  sugars." 

3.  The  property  that  glucose  and  other  sugars  have  of  dissolving  cupric 
hydrate  is  common  to  a  large  number  of  organic  compounds. 

67.  Boil  about  3  c.c.  of  Fehling's  solution  (see  Note  l)  in  a 
test  tube.  No  change  occurs.  Add  about  3  c.c.  of  the  glucose 
solution  and  boil  again.  A  red  precipitate  of  cuprous  oxide  is 
formed.  (Fehling's  test.) 

NOTES — 1.     Fehling's  fluid  is  prepared  as  follows  : 

(a)  Dissolve    103 '92  grams,  of  pure  copper   sulphate  in  warm  water  and 
dilute  to  one  litre. 


36  THE     CARBOHYDRATES.  [CH.    II. 

(6)  Dissolve  320  grams,  of  potassium  sodium  tartrate  (Rochelle  salt)  in 
warm  water,  add  a  little  carbolic  acid  to  prevent  the  growth  of  fungi,  dilute  to 
exactly  a  litre  and  filter. 

(c)  Dissolve  150  grams,  of  sodium  hydoxide  in  distilled  water  and  dilute 
to  1  litre. 

For  use  take  exactly  equal  quantities  of  a,  b,  and  c,  and  mix.  Though  the 
individual  constituents  keep  indefinitely,  the  fluid  when  prepared  suffers 
decomposition,  so  that  a  reduction  occurs  on  boiling.  For  this  reason  the  fluid 
should  be  prepared  just  before  use,  and  must  always  be  tested  by  boiling  before 
being  used. 

The  fluid  is  of  such  a  strength  that  the  copper  sulphate  in  10  c.c.  is  just 
reduced  by  '05  grams  of  dextrose. 

2.  The  addition  of  the  Rochelle  salt  is  for  the  purpose  of  dissolving  the 
cupric  hydoxide  that  would  otherwise  be  precipitated  by  mixing  (a]  and  (c) . 

3.  The  test  is  much  more  delicate  and  certain  than  Trommer's  test,  and 
should  always  be  used  in  preference  to  it. 

4.  If  the  fluid  that  is  being  tested  is  acid,  it  should  be  neutralised. 

5.  Ammonium   salts  considerably  interfere  with  Fehling's  test.     If  they 
are  present  a  little  extra  alkali  should  be  added,  and  the  mixture  boiled  for  two 
or  three  minutes  to  allow  of  the  evolution  of  the  ammonia. 

6.  In  testing  for  small  amounts  of  glucose  it  is  advisable  to  avoid  an  excess 
of   Fehling's    solution,   owing   to   the  excess  of  alkali  tending   to  destroy  the 
glucose  before  the  latter  can  exert  its  reducing  reaction  on  the  copper.     The 
neutral  solution  should  be  made  faintly  blue  with  Fehling's  solution,  and  then 
boiled. 

68.  To  5  c.c.  of  Benedict's  solution  in  a  test  tube,  add  about 
eight  drops  of  the  sugar  solution.  Boil  vigorously  for  one  or  two 
minutes  and  allow  the  tube  to  cool  spontaneously.  The  entire  body 
of  solution  will  be  rilled  with  a  precipitate,  red,  yellow,  or  green  in 
colour  depending  on  the  concentration  of  the  sugar.  (Benedict's 
test.) 

NOTES — 1.  Preparation  of  Benedict's  solution  for  qualitative  test. 
Dissolve  173  grams,  of  sodium  citrate  and  100  grams,  of  anhydrous  sodium 
carbonate  in  about  600  c.c.  of  distilled  water  by  the  aid  of  heat.  Pour  through 
a  folded  filter  and  make  up  to  850  c.c.  Dissolve  17'3  grams,  of  crystallised 
copper  sulphate  in  100  c.c.  of  water  and  make  up  to  150  c.c.  Pour  the 
carbonate-citrate  solution  into  a  large  beaker  and  add  the  copper,  solution 
slowly,  with  constant  stirring.  The  mixed  solution  is  ready  for  use  and  does 
not  deteriorate  on  long  standing. 

2.  Benedict's  solution  has  certain  advantages  over  Fehling's.  For 
example,  it  is  not  so  readily  reduced  by  uric  acid  or  urates,  nor  by  creatinine. 
It  is  not  reduced  by  chloroform,  which  is  sometimes  added  to  urine  as  a 
preservative.  It  does  not  destroy  a  small  amount  of  sugar,  as  Fehling's  does 


CH.    II.]  GLUCOSE.  37 

(see  note  6  to  previous  exercise) .  Also  it  can  be  used  for  testing  urines  for  sugar 
in  artificial  light,  since  it  is  the  bulk  and  not  the  colour  of  the  precipitate  that 
is  of  importance. 

69.  Boil  some  freshly  prepared  Barfoed's  reagent  and  add  to  it 
the  sugar  solution,  drop  by   drop,  boiling  the  whole  time.     A  red 
precipitate  of  cuprous  oxide  is  formed,  either  at  once,  or  on  stand- 
ing for  a  few  minutes.     (Barfoed's  test.) 

NOTES. — 1.  The  reagent  is  prepared  by  dissolving  66  gm.  cupric  acetate 
and  10  c.c.  of  glacial  acetic  acid  in  water  and  making  up  to  1  litre. 

2.  This  test  is  only  given  by  the  monosaccharides,  not  by  maltose  and 
lactose. 

3.  The   reagent   must   be   freshly   prepared,  otherwise   it  is  reduced  by 
maltose  and  lactose. 

4.  Chlorides  interfere  with  the  test,  causing  the  appearance  of  a  greenish 
white  precipitate. 

70.  Boil  1  part  of  Nylander's  solution  with  5   parts  of  the 
sugar    for    about   three   minutes   and   allow    to    cool.      A    black 
precipitate  of  metallic  bismuth  settles  out.     (Nylander's  test.) 

NOTES. — 1.  Nylander's  reagent  is  prepared  by  dissolving  50  gms.  of 
Rochelle  salt  and  20  gms.  of  bismuth  subnitrate  in  1  litre  of  8  per  cent,  caustic 
soda. 

2.  The  reaction  is  of  importance  in  detecting  small  quantities  of  glucose 
in  urine.  The  uric  acid  and  creatinine  of  concentrated  urine  reduce  Fehling's 
solution,  but  have  no  action  on  Nylander's  solution. 

71.  Treat  2  c.c.  of  a  '1  per  cent,  solution  of  safranine  with 
2    c.c.    of   the  glucose  solution  and  2  c.c.  of  5  per  cent,  sodium 
hydroxide.     Mix  and  boil,  avoiding  any  shaking.     The  opaque  red 
colour  gives  place  to  a  light  yellow,  owing  to  the   reduction  of  the 
safranine  to  a  "  leuco-base." 

72.  Add  to  the  solution  of  glucose  some  sulphindigotate  of 
soda  and  some  Na2CO3  and  boil.     The  blue  colour  turns  green, 
purplish,  red,  and  finally  yellow.     Shake  with  air :  the  blue  colour 
reappears.     (Mulder's  test.) 

NOTE. — These  two  experiments  illustrate  the  reducing  properties  of  glucose 
in  hot  alkaline  solution.  The  avidity  of  the  reduced  leuco-bases  for  oxygen  is 
shown  by  the  reappearance  of  the  colour  on  cooling  and  shaking  with  air. 


38  THE     CARBOHYDRATES.  [CH.    II. 

73,  Take  10  c.c.  of  a  1-5  per  cent,  solution  of  glucose  in  a 
test  tube.  Add  as  much  solid  phenyl-hydrazine  hydrochloride  as 
will  lie  on  a  sixpenny  piece,  and  at  least  twice  this  amount  of  solid 
sodium  acetate.  Dissolve  by  wTarming,  mix  thoroughly,  and  filter 
into  a  clean  test  tube.  Place  this  in  a  beaker  of  boiling  water  for 
at  least  half-an-hour,  keeping  the  water  boiling  the  whole  time. 
Set  the  tube  aside  to  cool  (do  not  cool  under  the  tap).  A  fine 
yellow  crystalline  precipitate  of  phenyl-glucosazone '  appears. 
Collect  some  of  this  by  means  of  a  pipette,  transfer  to  a  slide, 
cover  with  a  glass  and  examine  under  both  powers  of  the 
microscope.  Note  the  characteristic  arrangement  of  the  fine 
yellow  needles  in  fan-shaped  aggregates,  sheaves  or  crosses.  Make 
a  drawing  of  the  crystals  in  the  space  provided  at  the  end  of  the 
book. 

NOTES.— Glucose  is  an  aldehyde,   and,   like   all   aldehydes 'and   ketones, 

•  forms   a   compound  with    phenyl-hydrazine.       But   this   phenyl-hydrazone  of 

glucose  is  very  soluble,  and  cannot  be  readily  separated.     However,   in  the 

presence  of  an  excess  of  phenyl-hydrazine  at  100yC.   an  insoluble  osazone  is 

formed. 

CHO  CH:N.NH.C6H5 

I  I 

CHOH  C  :  N.NH.CeHr. 

+  3  C6H5.NH.NH2  -       | 
(CHOH)3  (CHOH),, 

I  I 

CH2OH  -  CH2OH 

Glucose  Phenyl-osazone    of    glucose 

(phenyl-glucosazone) 
+  2H2O  +  NH:i  +  C6H5.NH2 
Aniline. 

2.  Phenyl-hydrazine  is  a  yellow  basic  liquid,  insoluble  in  water,  but  soluble 
in  dilute  acids  to  form  salts.     If  the  base  itself  is  used,   two  or  three  drops 
should  be  dissolved  in  a  few  drops  of  strong  acetic  acid,  and  added  to  the  sugar 
solution. 

3.  Phenyl-hydrazine   hydrochloride,    C6Hr,.NH.NH2.HCl    does   not   give 
an  osazone  when  boiled  with  glucose,  unless  an  excess  of  sodium  acetate  be 
added.     This  acts  on  the  hydrochloride  to  form  phenyl-hydrazine  acetate  and 
sodium  chloride. 

B.     The  Disaccharides.   . 

These  carbohydrates  have  the  empirical  formula, 
C^H^On.  They  are  hydrolysed  by  boiling  with  dilute 


CH.    II.]  CANE-SUGAR.  39 

acids  or  by  the  action  of  certain  specific  enzymes  into  two 
molecules  of  monosaccharide. 

C12H220U  +  H20  =  C6H1206  +  C6H1206, 

The    three    disaccharides    of    physiological    interest    are 
cane-sugar,  maltose  and  lactose  (milk-sugar). 

Cane-sugar  (sucrose)  is  widely  distributed  in  the 
vegetable  kingdom,  where  it  functions  as  a  reserve 
material.  It  crystallises  well,  is  very  soluble  in  water, 
and  has  a  much  sweeter  taste  than  glucose. 

It  does  not  reduce  Fehling's  solution,  does  not  form 
an  osazone,  and  does  not  behave  as  an  aldelyde  or  ketone. 
It  is  hydrolysed  very  readily  by  boiling  acids  to  a  mixture 
of  glucose  and  fructose.  Cane-sugar  is  dextrorotatory, 
but  since  fructose  is  more  laevorotatory  than  glucose  is 
dextrorotatory,  a  mixture  of  the  two  in  equal  parts  is 
laevorotatory.  So  the  sign  of  rotation  being  inverted  by 
hydrolysis,  the  process  is  known  as  inversion,  and  the 
product  as  "  invert  sugar."  This  hydrolysis  is  also  effected 
by  the  enzyme  invertase  (sucrase),  which  is  found  in  the 
small  intestine  and  in  certain  yeasts. 

The  constitution  of  cane-sugar  is  not  yet  definitely 
established,  but  in  all  probability  it  is  formed  by  the 
condensation  of  glucose  and  fructose  in  such  a  way  as  to 
destroy  both  the  aldehyde  and  the  ketone  groups. 

O 


CH2(OH).C.(CH.OH)2.CH.CH2OH 

/ 
O 

CH.(CH.OH)2.CH.CH.OH.CH2.OH 

\ 

O 


40  THE      CARBOHYDRATES.  LCH.    II. 

74.  Repeat  experiments  65,  67  and  68,  with  a  freshly  prepared 
1  per  cent,  solution  of  pure  white  crystalline  cane-sugar  ("  coffee 
sugar").       Note    that    it    is    unaffected    by    alkali    and    exerts    no 
reducing  reaction  on  Fehling's  solution. 

75.  Treat    3    c.c.   of   the    solution  with  one  drop    of    strong 
sulphuric    acid    and    boil    for   a   minute.      Add   a    drop    of    litmus 
solution  and  neutralise  with  caustic  soda.     Apply   Trommer's   or 
Fehling's  test  to  portions  of  this  fluid.     A  well  marked  reduction  is 
obtained  in  both  cases. 

NOTE. — This  reaction  depends  on  the  fact  that  although  cane-sugar  is 
.a  non-reducing  sugar,  it  is  converted  to  equal  parts  of  glucose  and  laevulose  by 
boiling  with  dilute  mineral  acids. 

CiaHaaOn  +  H2O  =  C6H12O6+  C6H12O6 
Cane-sugar.  Glucose.     Laevulose. 

76.  Treat  four  drops  of  the  solution  of  cane-sugar  with  four 
•drops  of  2  per  cent,  solution  of  alpha-naphthol  in  alcohol  and  5  c.c. 
of   fuming  hydrochloric  acid.     Heat  to   boiling  point.     The   fluid 
immediately  begins  to  assume  a  rich  purple  tint. 

NOTES. — 1.  This  reaction  depends  on  the  fact  that  the  laevulose,  which 
is  formed  by  the  action  of  the  acid  on  the  cane-sugar,  yields  furfurol 
(furfuraldehyde) 


HC CH 


HC 


C.  CHO 


O 

which  in  its  turn  reacts  with  the  alpha-naphthol  to  give  a  purple  colour. 

2.  Glucose,  lactose,  and  maltose  only  give  this  reaction  very  feebly.     The 
polysaccharides  and  especially  cellulose  give  a  fair  reaction.     It  is  also  given  by 
certain  proteins,  when  it  is  known  as  Molisch's  rection. 

3.  In  using   the   reaction  as  a   test  for  cane-sugar,   great  care  must  be 
taken  to  remove  proteins  and  dextrins  from  solution  by  the  method  described  in 
Ex.  55.     The  residue  left  after  evaporation  of  the  alcohol  will  contain  all  the 
sugars  present  in  the  original  fluid. 

4.  Thymol  can  be  used  instead  of  alpha-naphthol. 

77.  Mix  a  solution  of  cane-sugar  with  one  of  glucose.  Boil 
the  mixture  with  Fehling's  solution,  adding  the  Fehling's  solution  to 
the  boiling  fluid  until  a  blue  colour  by  transmitted  light  indicates  a 
slight  excess  of  Fehling's  solution.  By  this  procedure  the  glucose 
is  destroyed,  but  the  cane-sugar  is  unaffected.  Filter  off  the 


CH.    II.]  MALTOSE.  41 

precipitate  of  cuprous  oxide.  Make  the  filtrate  acid  with  sulphuric 
and  boil.  Neutralise  the  solution,  add  a  Itttle  more  Fehling's  and 
boil  again.  A  well-marked  reduction  is  obtained  due  to  the 
production  of  glucose  and  laevulose  by  "  inversion  "  of  the  cane- 
sugar  by  the  acid. 

NOTE. — In  using  this  as  a  test  for  cane-sugar  in  the  presence  of  glucose,  the 
presence  of  the  polysaccharides  must  be  excluded  by  alcoholic  extraction  if 
necessary  :  and  the  solution  must  give  a  well-marked  alpha-naphthol  test,  as 
lactose  and  maltose,  after  boiling  with  Fehling's  solution,  give  a  reducing 
substance  by  acid  hydrolysis. 

78-  Seliwanoff  S  test  for  laevulose.  Obtain  a  neutral  solution 
containing  laevulose  as  in  Ex.  75.  To  5  c.c.  of  SeliwanofFs  re- 
agent add  a  few  drops  of  the  sugar  and  heat  the  solution  to  boiling. 
A  red  colouration  and  a  red  precipitate  are  formed.  The  precipitate 

dissolves  in  alcohol,  to  which  it  imparts  a  striking  red  colour. 

NOTES. — The  reagent  is  prepared  by  dissolving  0'05  grm.  of  resorcin 
in  100  c.c.  of  1  in  2  hydrochloric  acid. 

The  test  is  also  given  by  the  monosaccharides  after  long  boiling,  but 
.a  precipitate  is  not  usually  formed. 

Maltose  is  the  disaccharide  formed  as  the  final  product 
of  the  hydrolysis  of  starch  by  the  enzyme  ptyalin.  It  is 
hydrolysed  by  boiling  acids,  and  by  the  enzyme  maltose 
of  the  small  intestine,  to  two  molecules  of  glucose.  It 
exhibits  well  -  marked  reducing  properties  towards 
Fehling's  and  Nylander's  solutions,  but  not  towards 
Barfoed's.  It  forms  an  osazone  with  phenyl-hydrazine 
acetate,  which  is  more  soluble  than  glucosazone  and 
which  melts  at  206°C.  Constitutionally  it  is  glucose 
a-glucoside. 

79.  Repeat  experiments  65  and  67  with  a  '2  per  cent,  solution 
of  maltose.     It  behaves  like  glucose. 

80.  Boil  with  Barfoed's  reagent.    No  reduction.    (Distinction 
from  glucose.) 

81.  Examine     microscopically    and    draw    the     crystals    of 
phenyl-maltosazone  that  have  been  prepared  by  the  demonstrator. 
Note  that  they  are  much  broader  than  the  crystals  of  glucosazone. 
Make  a   drawing  of  the  crystals  in  the  space  provided  at  the  end  of 
the  book. 


42  THE     CARBOHYDRATES.  [CH.    II. 

Lactose  is  the  sugar  found  in  milk,  and  often  in  the 
urine  of  women  during  lactation.  It  has  reactions  very 
similar  to  those  of  maltose.  It  is  hydrolysed  by  boiling 
acids,  and  by  the  ferment  lactase  into  a  molecule  of 
glucose  and  one  of  galactose. 

Constitutionally  it  is  glucose-/?-galactoside. 

It  is  not  fermented  by  ordinary  yeast. 

The  osazone  melts  at  200°C. 

82.  Repeat  Exs.  65  and  67  with  a  -2  per  cent,  solution  of 
lactose.     It  behaves  like  glucose. 

83.  Boil  with  Barfoed's  reagent.    No  reduction.    (Distinction 
from  glucose.) 

84.  Examine     microscopically    and    draw    the     crystals     of 
phenyl-lactosazone  that  have  been  prepared  by  the  demonstrator. 
Notice  that  they  differ  considerably  from  glucosazone",-  separating, 
usually,   as  ovoid   or  spherical  clusters  of   fine  needles.     Make  a 
drawing  of  the  crystals  in  the  space  provided  at  the   end  of  the 
book. 

C.     The   Polysaccharides. 

These  compounds  are  formed  by  the  condensation  of 
more  than  two  molecules  of  monosaccharides.  Their 
general  formula  is  (C6H10O5)n. 

Starch  is  widely  found  in  the  vegetable  kingdom  as  a 
reserve  carbohydrate.  It  occurs  in  the  form  of  grains, 
the  form  of  which  is  characteristic  for  a  particular  plant. 
These  grains  may  consist  of  two  materials,  starch 
"granulose  "  and  starch  "cellulose",  the  latter  forming  a 
dense  envelope  to  the  grain ;  owing  perhaps  to  this  the 
grains  are  insoluble  in  cold  water,  and  are  only  slowly 
attacked  by  enzymes.  But  on  being  boiled  they  absorb 
water  and  swell  up  to  form  a  paste  that  is  readily 
attacked  by  certain  enzymes. 

Starch  has  a  very  high  molecular  weight,  and  on  being 
boiled  with  water  forms  an  opalescent  "  solution  "  that  is 
really  a  colloidal  suspension.  It  does  not  diffuse  through 


CH.    II. 


STARCH. 


43 


membranes  and  does  not  depress  the  freezing  point  of 
water. 

This  so-called  "  starch  paste  "  is  completely  precipi- 
tated by  half-saturation  with  ammonium  sulphate  and 
by  the  addition  of  an  equal  volume  of  strong  alcohol. 

The  most  characteristic  reaction  of  starch  is  the  blue 
colour  it  gives  with  free  iodine  solution.  It  does  not 
reduce  Fehling's  solution  and  is  only  slowly  affected  by 
boiling  alkalies. 

Starch  paste  is  hydrolysed  by  boiling  acids  and  by 
certain  enzymes,  which  are  therefore  called  the  amylolytic 
enzymes.  These  are  ptyalin  of  saliva,  amylopsin  of 
pancreatic  juice  and  the  diastases  found  in  malt  and 
certain  yeasts  and  moulds. 

The   products    of  hydrolysis    of    starch    by    such    a 
ferment  as  ptyalin  are  very  numerous.      The  following 
scheme  indicates  the  probable  course  of  the  hydrolysis, 
but  it  is  not  claimed  that  it  is  yet  finally  established. 
Starch  paste 

Soluble  Starch  (Amylodextrin) 


E1  trfU  nnrlov  f  r>!  n    I                                       Ma  1  1nHpX  trifl 

>  maltose. 

Eythrodextrin  II                    Maltodextrin  — 

|                                                  i 

>  maltose. 
>  maltose. 

Achroodextrin                        Maltodextrin  — 

1 

Achroodextrin                        Maltodextrin  — 

Stable  dextrin 


"1 
Maltodextrin- 


->  maltose. 


Maltose. 


I 
Glucose. 


44  THE     CARBOHYDRATES.  [CH.    II. 

85.  Place  a  small  amount  of  dry  potato-starch  on  a  slide,  add 
a  drop  of  water,  cover  with  a  slip  and  examine  under  the  microscope. 
Note  the  characteristic  oval  starch  grains,  the  concentric  markings 
and  the  hilum,  usually  eccentric.     Make  a  drawing  of  the  grains. 
Run  a  drop  of  iodine  under  the  slip ;  note  that  the  grains  take  on  a 
blue  colour. 

86.  Shake  a  small  amount  of  potato  starch  with  cold  water. 
The    starch    does  not  dissolve.     Filter,  and  add  a  drop  of  iodine 
solution    to   the  filtrate.     The    characteristic    blue    reaction  is  not 
obtained. 

87.  Shake    some    dry  starch  with  a  little  sodium    carbonate 
solution.      No   change  is  effected.      Repeat,  with    a  little   sodium 
hydoxide.    The  starch  is  immediately  gelatinised.    Add  a  few  drops 
of   iodine  solution,    a    blue  colour   is    not    obtained.    '-Treat   with 
strong  acetic  acid.     A  deep  blue  colour  appears. 

NOTE.  Free  iodine  is  necessary  to  give  the  blue  adsorption  compound  with 
starch.  Sodium  hydoxide  removes  free  iodine,  converting  it  into  iodide  and  iodate- 
The  action  of  the  acid  on  the  latter  causes  the  appearance  of  free  iodine  and  the 
blue  colour.  Always  neutralise  an  alkaline  solution  before  testing  for  the 
poly  sacchar  ides. 

88.  Take  as  much  starch  as  will  lie  on  a  shilling,  shake  it  up 
with  5  c.c.  of  water,  and  pour  into  100  c.c.  of  boiling  water,  stirring 
the    mixture    during   the   addition.     Boil    for   two    minutes.     The 
starch  becomes  gelatinised,  and  forms  a  thin,  somewhat  opalescent 
paste.     Cool    a   portion    under   the  tap  and  add  a  drop  of  iodine 
solution.     A  deep  blue  colour  is  formed. 

89.  Treat  5  c.c.  of  the  cold  starch  paste  with  an  equal  bulk  of 
saturated  ammonium  sulphate.     Shake  the  test  tube  and  allow  it  to 
stand  for  five  minutes.     The  starch  is  precipitated.     Filter  through 
a  dry  paper,  and  add  a  drop  of  iodine  solution  to  the  filtrate.     No 
blue  colour,  or  only  the  very  slightest  tint  is  obtained,  showing  that 
the   whole   of  the  starch   paste   is  precipitated   by  half -saturation 
with  (NH4)2S04. 

90.  Boil  5  c.c.  of  the  starch  paste  with  two  drops  of  concen- 
trated sulphuric  acid  for  about  15  seconds.     Note  that  the  solution 


CH.    II.]  DEXTRINS.  45 

becomes  perfectly  clear  and  translucent.  Add  two  drops  of  strong 
ammonia  to  neutralise  the  acid,  cool  under  the  tap,  add  an  exactly 
equal  bulk  of  saturated  (NH4)2SO4,  shake  the  tube  vigorously  and 
allow  it  to  stand  for  five  minutes.  Filter  through  a  dry  filter-paper 
and  add  two  drops  of  iodine  solution  to  the  filtrate.  A  deep  blue 
colour  is  obtained. 

NOTE. — Starch  paste  is  rapidly  converted  into  "  soluble  starch  "  on  boiling 
with  dilute  mineral  acids.  Soluble  starch  differs  from  starch  paste  in  that  it  is 
not  completely  precipitated  by  half-saturation  with  (NH^SC^  in  the  course  of 
five  minutes.  If  it  be  allowed  to  stand  for  twenty-four  hours,  however,  it  is 
completely  precipitated. 

91.  Take  10  c.c.  of  the  starch  paste  in  a  small  beaker.  Add 
five  drops  of  concentrated  sulphuric  acid,  bring  the  mixture  to  the 
boiling  point,  and  keep  it  boiling  for  seven  minutes.  Add  a  drop 
of  litmus  solution  and  neutralise  with  sodium  hydroxide,  keeping  the 
reaction  on  the  acid  rather  than  on  the  alkaline  side.  Cool  one  portion 
under  the  tap  and  add  a  drop  of  iodine  solution.  A  purple,  red  or 
brown  reaction  of  erythro-dextrin  is  obtained,  instead  of  the  original 
blue  reaction  of  starch.  To  the  other  portion  add  3  c.c.  of  Fehling's 
solution  and  boil.  A  well  marked  reduction  is  obtained. 

NOTE. — Starch  is  converted  to  erythro-dextrin  and  glucose  by  boiling  with 
dilute  mineral  acids.  If  the  boiling  is  prolonged  the  erythro-dextrin  is  converted 
into  glucose.  The  extent  of  boiling  required  to  destroy  the  whole  of  the  starch, 
and  yet  to  leave  some  erythro-dextrin  varies  with  the  concentrations  of  the 
starch  paste  and  of  the  acid  employed. 

The  Dextrins  are  polysaccharides  formed  by  the  partial 
hydrolysis  of  starch.  They  differ  a  great  deal  in  com- 
plexity, and,  with  the  exception  of  the  erythrodextrins,. 
are  characterised  and  separated  as  individuals  with 
considerable  difficulty. 

They  all  dissolve  in  water  to  form  a  clear  solution 
(distinction  from  glycogen).  They  are  insoluble  in  strong 
alcohol  and  in  ether.  They  all  reduce  Fehling's  solution 
with  the  exception  of  amylodextrin.  This  indicates  that 
they  contain  an  aldehyde  or  a  ketone  group  in  the 


46  THE     CARBOHYDRATES.  [CH.'II. 

molecule.  But  owing  to  the  large  size  of  the  molecule  the 
reducing  power  of  the  higher  dextrin s  is  very  slight. 

Only  the  higher  members  yield  a  colour  with  iodine. 

Amylodextrin  gives  a  pure  blue  with  iodine.  It  is 
slowly  precipitated  by  half-saturation  with  ammonium 
sulphate :  immediately  by  full  saturation. 

Erythrodextrin  I.  gives  a  purplish  colour  with  iodine : 
is  precipitated  by  full  saturation  with  ammonium  or 
magnesium  sulphates. 

Erythrodextrin  II.  gives  a  red  colour  with  iodine.  It 
is  precipitated  by  full  saturation  with  ammonium,  but 
not  by  magnesium  sulphate. 

Erythrodextrin  III.  gives  a  red  brown  colour,  and  is 
not  precipitated  by  any  mixture  of  salts. 

Achrodextrins  give  no  colour  with  iodine,  and  are  not 
precipitated  by  salts. 

Maltodextrin  is  the  name  given  to  a  substance  that 
was  separated  from  the  mixed  products  obtained  by  the 
hydrolysis  of  starch  paste  by  malt  diastase.  It  consists 
of  three  molecules  of  maltose  united  together  with  the 
elimination  of  two  molecules  of  water,  and  retaining  a 
terminal  aldehyde  group.  It  reduces  Fehling's  solution, 
but  does  not  ferment  with  yeast  or  give  an  osazone.  It 
is  hydrolysed  by  ferments  very  rapidly  to  maltose  :  by 
acids  to  glucose. 

Stable  dextrin  is  also  formed  by  the  action  of  arnylolytic 
enzymes  on  starch  paste.  It  is  rather  resistant  to  the 
action  of  the  enzymes,  but  is  slowly  converted  into  a 
mixture  of  equal  parts  of  maltose  and  glucose.  It  is 
formed  by  the  condensation  of  forty  molecules  of  glucose 
with  the  elimination  of  thirty-nine  molecules  of  water. 
In  the  hydrolysis  of  starch  by  enzymes,  about  80  per  cent. 


CH.    II.]  DEXTRINS.  47 

of  the  starch  is  converted  into  maltose,  the  remaining  20 
per  cent,  being  stable  dextrin. 

The  Dextrins. 

92.  Shake  a  little  commercial  dextrin  with  some  cold  water. 
An  opalescent  solution  is  formed.     Boil  the  solution.     It  becomes 
perfectly  translucent.     (Distinction  from  glycogen.) 

Use  a  3  per  cent,  solution  of  commercial  dextrin  for  the  follow- 
ing reactions: 

93.  To  about  5  c.c.  of  the  dextrin  solution  add  iodine  solution, 
drop  by  drop,  noting  the  colour  at  every  addition.     The  colour  is  at 
first  almost  a  pure  blue  but  it  changes  through  a  rich  purple-red  to 
a  red -brown  as  the  iodine  is  added. 

94.  Repeat  the  above  experiment,  but  boil  and  then  cool  the 
tube  after  each  addition.      The  colour  disappears  on  boiling,  but 
does  not  reappear  on  cooling  until  several  drops  of  iodine  have  been 
added. 

95.  Add  a  drop  or  two  of  the  starch  paste  prepared  in  Ex.  88 
to  about  5  c.c.  of  the  dextrin  solution.     To  this  mixture  add  diluted 
iodine  solution,  drop  by  drop.     The  first  additions  produce  a  pure 
blue  colour,  and  it  is  not  till  a  considerable  amount  of  iodine  has 
been  added  that  the  solution  acquires  a  purplish  tint. 

NOTE. — The  affinity  of  starch  for  iodine  is  so  much  greater  than  that  of 
dextrin  that  the  characteristic  colour  reactions  of  ery  thro-dextrin  are  not  obtained 
until  all  the  starch  has  been  saturated  with  iodine.  Even  then  it  is  sometimes 
difficult  to  detect,  owing  to  the  deep  blue  starch  reaction. 

96.  Treat  5  c.c.  of  the  dextrin  solution  with  about   10  drops 
of  the  starch  paste :  to  the  mixture  add  an  equal  bulk  of  saturated 
(NH4)2SO4,  shake  vigorously,  and  allow  to  stand  for  five  minutes. 
The  starch  is  precipitated.     Filter  through  a  dry  paper,  and  to  a 
portion  of  the  filtrate  add  a  drop  or  two  of  iodine  solution.     The 
purple  red  reaction  of  erythro-dextrin  is  obtained. 

97.  Saturate  5  c.c.  of  the  dextrin  solution  by  boiling  with  an 
-excess  of  finely  powdered  ammonium  sulphate.     Note  the  precipitate 
of   erythro-dextrin  produced.     Cool  under  the  tap  and  filter.     To 
the  filtrate  add  a  drop  of  iodine.     A  red-brown  colour  is  produced. 


48  THE     CARBOHYDRATES.  [CH.    II. 

NOTE. — This  colour  is  due  to  the  fact  that  erythro-dextrin  III.  is  not 
precipitated  by  ammonium  sulphate.  This  is  the  method  employed  for  the 
identification  of  erythro-dextrin  in  the  presence  of  glycogen,  which  is  completely 
precipitated  by  saturation  with  ammonium  sulphate. 

98.  Boil  a  few   c.c.    of   the    dextrin    solution    with   a  small 
amount    of    Fehling's   fluid.     A  well-marked  reduction  is   usually 
obtained. 

NOTE. — Commercial  dextrin  is  generally  prepared  by  the  action  of  dilute 
acids  on  starch  (See  Exercises  90  and  91),  the  action  being  stopped  as  soon  as 
a  portion  fails  to  give  a  blue  colour  with  iodine,  and  the  products  then  being 
precipitated  by  alcohol.  Such  preparations  contain  some  dextrose,  and  often  a 
little  soluble  starch.  At  the  same  time  it  must  be  noted  that  the  achroo-dextrins 
have  a  reducing  action  themselves  even  when  thoroughly  separated  from  the 
dextrose. 

99.  Take  10  c.c.  of  the  dextrin  solution  in  a  small  flask  ;  add 
30  c.c.  of  strong  (95  per  cent.)   alcohol,  place  the  thumb  over  the 
mouth  of  the  flask  and  shake  vigorously  for  some  seconds.     Note 
that  a  portion  of  the  dextrin  is  precipitated  as  a  gummy  mass  which 
sticks  to  the  sides  of  the  flask. 

Pour  off  the  alcohol,  filter  it  and  label  the  filtrate  A.  Rinse 
the  flask  out  with  a  few  c.c.  of  alcohol,  shake  off  as  much  of  this 
alcohol  as  possible,  and  add  10  c.c.  of  hot  water.  Shake  this  round 
the  flask  till  the  whole  of  the  gummy  precipitate  dissolves.  Divide 
the  solution  into  three  portions,  B,  C,  and  D.  To  B  add  a  drop 
of  iodine :  a  purple  colour  is  produced.  Boil  C  with  a  little 
Fehling's  solution :  only  a  slight  reduction  takes  place.  Boil  D 
with  two  drops  of  concentrated  sulphuric  acid  for  two  minutes, 
neutralise  with  NaOH,  and  boil  with  a  little  Fehling's  solution :  a 

C 

well-marked  reduction  occurs. 

100.  To  a  portion  of  filtrate  A,  add  a  drop  of  iodine  solution. 
No  colour  is  produced.     To  another  portion  of  about  5  c.c.  add  an 
equal  bulk  of  strong  alcohol.     A  white  precipitate  of  achroo-dextrin 
is  formed. 

Glycogen  is  a  reserve  polysaccharide  found  in  the  liver 
and  muscles.  It  forms  a  white  amorphous  powder, 
soluble  in  water  to  form  an  opalescent  solution.  It  is 


CH.    II.]  GLYCOGEN.  49 

precipitated  from  solution  by  the  addition  of  an  equal 
volume  of  strong  alcohol  or  by  full  saturation  with 
ammonium  sulphate.  It  does  not  reduce  Fehling's 
solution,  form  an  osazone  or  ferment  with  yeast.  It 
gives  a  reddish  colour  with  iodine.  By  boiling  acids  it 
is  hydro]ysed  to  glucose  :  by  most  of  the  diastatic  enzymes 
to  maltose,  but  by  the  diastase  found  in  the  liver  to 
glucose.  It  is  not  affected  by  boiling  alkalies.  It  is 
dextro-rotatory. 

Estimation.  Pfliiger's  method  is  undoubtedly  the  best.  20  to  lOOgm. 
of  the  tissue  is  cut  into  small  pieces  and  placed  in  an  Erlenmeyer  flask  of 
Jena  glass.  100  c.c.  of  60%  potash  ("  pure  by  alcohol " — sp.  gr.  1.  438)  is 
added,  a  reflux  condenser  fitted,  and  the  flask  immersed  for  three  hours  in 
a  boiling  water  bath.  The  alkali  destroys  the  proteins  without  attacking 

the  glycogen. 

/ 
After  cooling  200  c.c.  of  water  and  800  c.c.  of  96%  alcohol  are  added 

and  the  mixture  left  to  stand  over  night.  The  glycogen  is  thus  precipi- 
tated free  from  protein.  The  supernatant  fluid  is  carefully  decanted  and 
filtered.  The  precipitate  is  washed  with  ten  times  its  volume  of  66% 
alcohol,  containing  1  c.c.  per  litre  of  saturated  sodium  chloride.  After 
settling,  the  fluid  is  filtered  through  the  original  filter  paper.  This 
process  is  repeated  once  more,  and  then  the  precipitate  is  shaken  with  ten 
times  its  volume  of  96%  alcohol  and  filtered  through  the  same  paper. 
The  precipitate  is  washed  with  ether,  dissolved  in  boiling  water  and  the 
solution  made  up  to  one  litre.  200  c.c.  of  this  are  treated  with  10  c.c.  of 
concentrated  HC1  and  heated  in  a  flask  on  a  boiling  water  bath  for  three 
hours,  to  convert  the  glycogen  into  glucose.  After  cooling,  the  solution  is 
neutralised  with  20%  potash  and  filtered  through  a  small  paper  into 
a  250  c.c.  measuring  flask.  The  washings  from  the  flask  used  for 
inversion  are  filtered  through  the  same  paper  to  remove  the  last  traces  of 
glucose,  and  the  solution  brought  up  to  250  c.c.  The  percentage  of  glucose 
in  the  solution  is  determined  by  analysis.  This  multiplied  by  -927  gives 
the  amount  of  glycogen  in  the  200  c.c.  of  the  solution  used  for  inversion, 
and  so  the  percentage  in  the  tissue  can  be  readily  calculated. 

Preparation.  A  rabbit,  which  has  had  a  full  meal  of  carrots  some  five 
or  six  hours  previously,  is  killed  by  decapitation.  The  liver  is  cut  out  as 
quickly  as  possible,  and  the  gall-bladder  removed.  The  liver  is  rapidly 
chopped  into  small  pieces,  a  small  portion  being  reserved  for  Ex.  106, 
and  the  remainder  immediately  thrown  into  boiling  water.  After  about 
two  minutes  boiling  the  larger  morsels  are  strained  off,  pounded  to  a  paste 
with  sand  in  a  mortar,  and  replaced  in  the  boiling  water.  The  proteins  in 
solution  are  then  coagulated  by  making  the  boiling  fluid  just  acid  with 
acetic  acid.  The  fluid  is  filtered  through  coarse  filter  paper.  In  this  way 
a  crude  solution  of  glycogen  is  obtained. 


50  THE     CARBOHYDRATES.  [CH.    II. 

101.  Boil  5  c.c.  in  a  test  tube.     The  characteristic  opalescence 
does  not  disappear.     (Distinction  from  erythro-dextrin.) 

102.  To  a  small  amount  of  the  coole'd  solution  add    iodine, 
drop    by    drop.      A    red   colour    is    formed,    which    disappears    on 
shaking,  until  with  a  certain  amount  of  iodine  added  it  is  permanent. 
Now    heat    the    solution.     The  colour   disappears,   to  reappear  on 
cooling. 

NOTE.— If  much  protein  is  present  in  solution  the  colour  will  not  reappear 
on  cooling  unless  a  considerable  amount  of  iodine  be  added.  This  is  due  to  the 
fact  that  proteins  combine  with  iodine  to  form  an  iodo-protein. 

103.  Saturate    10  c.c.    of    the    solution    with    finely -powdered 
(NHjaSO*.     The  glycogen  is  precipitated.     Filter,  and  add  a  drop 
or  two  of  iodine  to  the  nitrate.     No  red  colour  is  produced.    Scrape 
the  precipitate  off  the  paper,  boil  with  a  small  amount  of  water. 
The  solution  is  markedly  opalescent.     Cool  the  solution,  and  add 
iodine.     A  port-wine  red  colour  is  obtained. 

104.  Boil  5  c.c.   of  the  solution  with  a  little  Fehling's  fluid. 
A  very  slight  or  no  reduction  is  obtained. 

NOTE. — If  the  liver  has  been  rapidly  boiled,  no  sugar  will  be  present.  If 
delay  has  occurred  during  the  initial  stages  of  the  preparation,  some  of  the 
glycogen  will  have  been  converted  into  glucose.  ,  (See  Exercise  106.) 

105.  To  10  c.c.  of  the  solution  add  20  c.c.  of  strong  alcohol, 
shake  vigorously  and  filter.     To  a  portion  of  the  filtrate  add  iodine 
solution.     No  colour  is  obtained,  showing  that   the  whole    of    the 
glycogen   is  precipitated.     Dissolve  the  precipitate   in   a   little  hot 
water :  note    that    it    is    opalescent.      Add   three    drops    of    strong 
sulphuric  acid  and  boil  for  about  three  minutes :  the  opalescence 
disappears.     Neutralise  with  sodium  hydroxide  and  apply  Fehling's 
test;     A    marked  reduction  occurs,   due  to   the   conversion   of  the 
glycogen  into  glucose  by  the  boiling  acid. 

106.  The  portion  of  rabbit's  liver  that  was  reserved  has  been 
kept  in  a  warm  place  for  about  six  hours  and  extracted  with  boiling 
water  as  before.     (Or  a  decoction  of  the  liver  of  a  sheep  obtained 
from  a  butcher  may  be  used.)     Note  that  the  solution  is    almost 


CH.    II.]  ESTIMATION    OF    SUGAR.  51 

translucent.  To  a  portion  add  iodine :  only  a  very  slight  or  no  red 
colour  at  all  is  produced.  To  another  portion  apply  Fehling's  test : 
a  well-marked  reduction  occurs. 

107.  Prepare  a  solution  which  contains  equal  quantities  of 
1  per  cent,  starch  paste  (freshly  prepared),  of  a  strong  solution  of 
glycogen  and  of  a  3  per  cent,  solution  of  commercial  dextrin.  Note 
that  the  mixture  is  markedly  opalescent. 

To  a  small  portion  add  diluted  iodine,  and  note  that  a  pure 
blue  starch  reaction  is  obtained. 

To  another  portion  of  about  5  c.c.  add  an  equal  bulk  of 
saturated  (NH^aSO*,  shake  vigorously,  leave  for  five  minutes,  and 
filter.  Note  that  a  portion  of  the  filtrate  gives  a  reddish  colour 
with  iodine,  and  that  it  is  distinctly  opalescent.  Indication  of  the 
presence  of  glycogen. 

Saturate  the  remainder  of  the  fluid '  with  finely-powdered 
(NH4)2SO4  and  filter.  The  filtrate  gives  a  reddish-brown  colour 
with  iodine.  Indication  of  the  presence  of  erythro-dextrin. 

D.     The  Quantitative  Estimation  of  Sugar. 

The  basis  of  nearly  all  the  modern  methods  for  the 
volumetric  estimation  of  the  sugars  is  the  determination 
of  the  amount  of  the  sugar  solution  necessary  to  reduce  a 
given  volume  of  Fehling's  solution.  The  chief  difficulty 
of  the  original  method  lies  in  deciding  the  exact  point 
when  the  copper  is  reduced,  as  indicated  by  the  complete 
disappearance  of  the  blue  colour.  This  is  obscured  by  the 
red  precipitate  of  cuprous  oxide  that  is  deposited. 

In  Ling's  method  an  indicator  is  used  to  determine 
this  point.  In  Pavy's  method  strong  ammonia  is  added 
to  form  a  soluble  cuprous  compound.  In  Benedict's 
method  potassium  sulphocyanide  is  employed  for  the 
same  purpose. 


52  THE     CARBOHYDRATES.  [CH.    II. 

Of  the  methods  given  below,  Bang's  is  undoubtedly 
the  most  accurate,  and  is  to  be  preferred  when  a  very 
reliable  estimation  of  sugar  is  required. 

As  a  standard  method  for  general  work  I  can  strongly 
recommend  Benedict's. 

Standardisation  of  the  Solutions.  Owing  to  the  fact  that  individual 
workers  go  to  rather  a  different  end  point,  it  is  advisable  to  perform 
estimations  of  a  standard  solution  of  sugar.  This  is  prepared  as  follows  : 
9*5  grams  of  pure  cane  sugar  are  dissolved  in  water  and  the  solution 
accurately  made  up  to  1000  c.c.  Of  this  solution  100  c.c.  are  boiled  with 

"NT 

30  c.c-  of  i-  HC1,  the   mixture  being  kept  boiling  for  one  minute.     It  is 

then  cooled,  neutralised  by  the  addition  of  30  c.c.  of  —  NaOH  and  made 

up  to  200  c.c.  with  water.     Such  a  solution  contains  0*5  gm.  of  invert 
sugar  per  cent. 

A  titration  of  Fehling's  or  Benedict's  solution  is  performed  with  this, 
and  the  result  noted.  Suppose  that  10  c.c.  Fehling's  solution  are  found  to 
be  reduced  by  0'054  gm.  of  invert  sugar,  use  this  factor  rather  than  the 
theoretical  0'05. 

108.     Benedict's  Method. 

Principle  of  the  Method. — An  alkaline  solution  of  copper 
sulphate,  containing  thiocyanate  is  boiled  and  the  sugar  solution 
run  in  from  a  burette  till  the  blue  colour  just  disappears.  The 
thiocyanate  forms  a  white  insoluble  compound  with  the  cuprous 
hydroxide  formed  by  the  reduction  of  the  copper,  and  so  there  is  no 
red  cuprous  oxide  precipitated  to  obscure  the  blue  tint.  A  little 
potassium  ferrocyanide  is  also  added  to  prevent  any  possibility  of 
the  deposition  of  the  cuprous  oxide. 

Preparation  of  the  Solution. — With  the  aid  of  heat  dissolve 
Sodium  citrate  ...  ...  200  grams. 

Sodium  carbonate  (cryst) 200  grams. 

(or  anhydrous  sod.  carb.  75  grams.) 
Potassium  thiocyanate  (sulphocyanide)        125  grams. 

in   enough  distilled  water  to  make  about  800   c.c.   of  the  mixture 
and  filter,  and  cool  to  room  temperature. 


CH.    II.] 


BENEDICTS    METHOD. 


53 


Dissolve  18  grams  of  pure,  air-dried  crystalline  copper 
sulphate  in  about  100  c.c.  of  distilled  water,  and  pour  it  slowly  into 
the  other  liquid  with  constant  stirring.  Add  5  c.c.  of  a  5%  solution 
of  potassium  ferrocyanide  and  then  distilled  water  to  make  the  total 
volume  1000  c.c.  The  solution  appears  to  keep  indefinitely, 
without  any  special  precaution,  such  as  exclusion  of  light,  etc. 

Method  of  Analysis. — Fit  a  4-oz.  flask  into  a  ring  of  a  retort 
stand  of  such  a  size  that  it  is  fairly  firmly  held.  There  is  no 
need  to  use  a  wire  gauze.  Arrange  the  flask  at 
such  a  height  over  a  Bunsen  burner  that  the 
reagent  can  be  kept  briskly  boiling  by  means  of  a 
small  flame.  In  the  flask  place  3  to  4  grams  of 
anhydrous  sodium  carbonate.  This  can  be  roughly 
measured  by  taking  a  depth  of  1  inch  in  a  dry  test 
tube.  Then  add  25  c.c.  of  the  reagent  and  heat 
till  the  carbonate  is  in  solution.  Run  the  sugar 
solution  in  from  a  burette,  which  is  best  held  in 
the  hand.  Run  the  sugar  in  at  a  fair  rate,  till  a 
bulky  chalk-white  precipitate  is  formed  and  the 
blue  colour  lessens  perceptibly  in  intensity. 
From  this  point  the  sugar  is  added  more  and 
more  slowly,  with  constant  vigorous  boiling,  until 
the  disappearance  of  the  last  trace  of  blue  colour, 
which  marks  the  end-point.  If  the  volume  of  the 
sugar  used  is  less  than  5  c.c.,  dilute  it  accurately 
with  water  till  about  10  c.c.  are  judged  necessary. 
Repeat  the  titration  with  this  as  before. 


Fig.  1. 


NOTES. — There  is  a  tendency  to  run  in  an  excess  of  the  sugar,  unless 
special  care  is  exercised  throughout  the  titration  and  particularly  at  the  end. 
The  solution  must  be  kept  vigorously  boiling  during  the  entire  process,  and 
towards  the  end  the  sugar  must  be  added  in  portions  of  a  drop  or  two,  with  an 
interval  of  about  30  seconds  after  each  addition.  Should  the  mixture  become 
too  concentrated,  boiled  distilled  water  may  be  added  to  replace  that  lost  by 
evaporation. 

The  titration  can  also  be  carried  out  in  a  white  porcelain  dish  of  10  to 
15^cm.  in  diameter. 

Should  the  solution  bump  excessively,  a  small  amount  of  powdered 
pumice  stone  may  be  added. 


54  THE     CARBOHYDRATES.  [CH.    II. 

Calculation  of  Results. 

25  c.c.  of  Benedict's  solution  are  reduced  by  0-05      grm.  of  glucose. 

0-053    grm.  laevulose. 
0-074    grm.  maltose. 
0-0676  grm.  lactose. 
Example. — First  titration  required  2-4  c.c. 

Solution  diluted  1  in  4  (10  c.c.  of  sugar  diluted  with  30  c.c.  water). 
Second  titration  required  9-7  c.c. 

So  9-7  c.c.  diluted  solution  contain  0-05  grm.  glucose. 

•05    x    100 


100  c.c.  diluted  solution  contain 
100  c.c.  original  solution  contain 


9-7 
0'05    x    100  x  4 


9-7 
Percentage  of  glucose  =  2-06. 

109.    Fehling's  Method. 

Preparation  of  solution.     See  Ex.  67,  p.  35. 

Method.  With  a  pipette  measure  10  c.c.  of  freshly  prepared 
Fehling's  solution  into  a  small  flask.  Add  40  c.c.  of  distilled  water, 
heat  the  mixture  till  it  boils  and  keep  it  boiling  the  whole  time. 
Run  in  the  sugar  solution  from  a  burette,  C'5  to  1  c.c.  at  a  time, 
allowing  the  mixture  to  boil  for  about  15  sees,  between  each 
addition.  A  red  precipitate  of  cuprous  oxide  forms  and  the 
intensity  of  the  blue  in  the  supernatant  fluid  decreases.  Continue 
to  add  the  sugar  till  this  is  completely  removed.  This  is  best 
determined  by  holding  the  flask  by  the  rim  at  the  neck  and  viewing 
it  by  transmitted  light.  If  an  excess  of  sugar  be  added  a  yellow  or 
brown  colour  appears  due  to  the  formation  of  caramel  by  the  action 
of  the  alkali  on  the  sugar. 

If  less  than  5  c.c.  of  the  sugar  are  used,  the  solution  must 
be  diluted  till  about  10  c.c.  are  necessary.  Thus  if  2'5  c.c.  are  used 
in  the  first  rough  titration,  the  sugar  should  be  diluted  1  in  4, 
by  taking  25  c.c.  and  adding  water  till  the  volume  of  the  solution  is 
100  c.c.  The  burette  is  washed  out  and  filled  with  this  diluted 
solution  and  the  process  repeated.  But  this  time  run  in  nearly  the 
whole  of  the  sugar  solution  judged  necessary  at  such  a  rate  that  the 
mixture  does  not  go  off  the  boil.  Then  add  O'l  to  0'2  c.c.  at  a  time 
till  the  reduction  is  complete.  This  titration  should  be  repeated  at 
least  once  more. 


CH.    II.]  LING'S    METHOD.  55 

Calculation.      lOc.c.  of  Fehling's  solution  are  reduced  by  O'Sgm.  glucose. 
Example.     1-5  c.c.  of  the  original  solution  necessary. 
Sugar  diluted  1  in  7  (10  c.c.  sugar  made  up  to  70  c.c.) 
10-2 c.c.  diluted  sugar  solution  required  for  10 c.c.  Fehling's. 
10-2  c.c.  dil.  sugar  =  -05  gm.  glucose. 

•05  X  100     , 

lOOc.c =  

10-2 

•05  x  100  x  7 
100  c.c.  original  sugar  =  

=  3-43  per  cent. 

110.     Ling's  Method. 

Preparation  of  the  indicator.  Dissolve  1*5  gm.  ammonium 
thiocyanate  and  1  gm.  ferrous  ammonium  sulphate  in  10  c.c.  water 
at  about  45°  C.  and  cool  at  once.  Add  2'5  c.c.  of  concentrated 
hydrochloric  acid.  The  solution  thus  obtained  has  invariably 
a  brownish-red  colour,  due  to  the  presence  of  some  ferric  salt.  Add 
zinc  dust,  in  small  portions  at  a  time,  till  the  fluid  is  just  colourless. 
On  standing  for  some  time  the  red  colour  reappears,  and  must  be 
removed  again  by  a  trace  of  zinc  dust.  But  the  delicacy  of  the 
indicator  is  impaired  by  being  decolourised  several  times.  When 
this  indicator  is  treated  with  a  cupric  salt,  the  colourless  ferrous 
thiocyanate  is  oxidised  to  the  red  ferric  thiocyanate. 

Method  of  analysis.  10  c.c.  of  Fehling's  solution  and  about 
30  c.c.  of  water  are  boiled  in  a  flask  and  the  sugar  solution  is  run  in 
from  a  burette  as  described  above  in  Fehling's  method.  The 
indicator  is  not  used  till  the  blue  colour  has  nearly  disappeared. 

Then  place  a  drop  of  the  indicator  on  a  white  slab.  Transfer 
a  drop  of  the  mixture  from  the  flask  to  the  middle  of  the  drop  of 
the  indicator  as  rapidly  as  possible  by  means  of  a  glass  tube.  If 
a  red  colour  appears  immediately  on  touching  the  drop  the 
reduction  is  not  completed.  More  sugar  must  be  added  and  a  fresh 
drop  of  the  indicator  used  as  before  till  no  colour  or  only  a  faint 
tinge  of  red  is  obtained.  If  less  than  5  c.c.  of  the  sugar  solution  are 
necessary  to  complete  the  reaction,  the  solution  must  be  diluted  till 
about  10  c.c.  are  required,  as  described  above  in  Fehling's  method. 

Special  precautions.  Use  a  glass  tube,  not  a  rod,  for  trans- 
ferring the  drop. 


56  THE     CARBOHYDRATES.  [CH.    II. 

Do  not  put  your  finger  on  the  top  of  the  tube.  Dip  it  in  the 
flask  and  transfer  it  immediately  to  the  indicator.  The  flask  may 
be  taken  off  the  boil  for  an  instant  while  this  is  done. 

Do  not  stir  the  drops  on  the  slab. 

Wash  the  tube  before  using  it  again. 

Calculation  of  results.  This  is  the  same  as  in  Fehling's 
method. 

111.    Bang's  Method. 

Principle.  A  known  volume  of  copper  thiocyanate 
in  potassium  carbonate  is  boiled  for  three  minutes  with  a 
given  volume  of  the  glucose  solution,  that  is  not  sufficient 
to  reduce  it  completely.  The  copper  in  excess  is  deter- 
mined by  titration  with  hydroxylamine  solution.  Both 
the  sugar  and  the  hydroxylamine  reduce  the  copper  to 
colourless  cuprous  thiocyanate,  so  the  end'  point  is 
readily  observed. 

Preparation  of  Solutions. 

1.  12*5  grams  of  copper  sulphate  are  dissolved  by  heat  in  75  c.c.  of 
water  and  the  solution  cooled  to  25  ;'C.     In  a  large  porcelain  basin  250 
grams    potassium  -carbonate,    200    grams  potassium  thiocyanate  and  50 
grams  potassium  bicarbonate  are  dissolved  by  stirring  in  600  c.c.   water. 
If  the  potassium  bicarbonate  does  not  dissolve  it  must  be  heated  on  the 
water  bath  to  40PC.,  but  no  higher.     It  is  then  cooled  to  15CC.  and  the 
copper  solution  mixed  with  it  in  small  quantities  at  a  time  with  frequent 
shaking,   to  prevent   any  large   amount  of  precipitate   forming.        The 
solution  is  then  made  up  to  1  litre. 

2.  6-55  grams  of  hydroxylamine  sulphate  or  5-56  grams  hydroxylamine 
chloride  are  dissolved  in  water  and  the  solution  added  to  one  of  200  grams 
potassium  thiocyanate  in  1500  c.c.  water.     The  volume  is  made  up  to  2 
litres. 

Method  of  estimation.  The  amount  of  glucose  added  must  be 
less  than  0*06  gm.  If,  therefore,  the  solution  contain  less  than  0*6 
per  cent.,  10  c.c.  of  it  are  taken  for  the  estimation.  If  it  contain 
more  than  this,  then  such  an  amount  must  be  taken  as  will  yield  a 
total  amount  less  than  O06  gm.  In  all  cases  the  sugar  solution 
must  be  made  up  to  10  c.c.  Where  there  is  no  previous  knowledge 
as  to  the  strength  of  the  sugar  solution  a  preliminary  titration 
should  be  made  by  boiling  10  c.c.  of  the  sugar  with  50  c.c.  of  the 
copper  solution  for  three  minutes.  If  the  blue  colour  disappears, 


CH.    II.] 


BANG'S  METHOD. 


57 


repeat  with   5   c.c.,  and  so  on  until  the  amount  is  found  that  does 
not  discharge  the  blue. 

Mix  the  10  c.c.  sugar  solution  with  50  c.c.  of  the  copper  solu- 
tion in  an  Erlenmeyer  flask.  Place  on  a  wire  gauze  over  a  Bunsen 
burner  and  bring  it  to  the  boil.  Maintain  the  boiling  for  exactly 
three  minutes.  Cool  the  solution  quickly  by  holding  the  flask 
under  the  cold  water  tap.  Titrate  with  the  hydroxylamine  solution 
from  a  burette,  running  it  in  rather  slowly  with  frequent  shaking 
so  as  to  prevent  any  precipitate  forming,  which  spoils  the  result. 
Add  the  hydroxylamine  until  the  blue  colour  is  discharged. 

Calculation  of  result.  The  greater  the  excess  of  copper  present,  the 
greater  is  the  reduction  caused  by  a  given  weight  of  glucose.  The  reduction  is 
therefore  not  proportional  to  the  amount  of  sugar  employed  in  the  determina- 
tion. A  table  has  been  prepared  showing  the  weight  of  glucose  corresponding 
to  the  amount  of  hydroxylamine  solution  that  is  necessary  to  decolourise  the 
unreduced  copper. 

Example.  2  c.c.  of  the  sugar  solution  and  8  c.c.  of  water  were  used. 
Volume  of  hydroxylamine  required  was  14'2  c.c.  The  table  shews  that 
37 '5  mg.  of  glucose  were  present. 

So  percentage  of  glucose  is  -0375  x  — -  =  1-875. 

Table  for  calculation  of  amount  of  glucose  from  hydroxylamine  used  in 
Bang's  method. 


Hydroxylamine 
solution  c.c. 

Glucose 
mgm. 

Hydroxylamine 
solution  c.c. 

Glucose 
mgm. 

Hydroxylamine 
solution  c.c. 

Glucose 
mgm. 

43-85 

5 

25-10 

24 

10-20 

43 

42-75 

6 

24-20 

25 

9-50 

44 

41-65 

7 

23-40 

26 

8-80 

45 

40-60 

8 

22-60 

27 

8-20 

46 

39-50 

9 

21-75 

28 

7-65 

47 

38-40 

10 

21-00 

29 

7-05 

48 

37-40 

11 

20-15 

30 

6-50 

49 

36-40 

12 

19-35 

31 

5-90 

50 

35-40 

13 

18-55 

32 

5-35 

51 

34-40 

14 

17-75 

33 

4-75 

52 

33-40 

15 

16-95 

34 

4-20 

53 

32-45 

16 

16-15 

35 

3-60 

54 

31-50 

17 

1535 

36 

3-05 

55 

30-55 

18 

14-60 

37 

2-60 

56 

29-60 

19 

13-80 

38 

2-15 

57 

28-65 

70 

13-05 

39 

1-65 

58 

27-75 

21 

12-30 

40 

1-20 

59 

26-85 

22 

11-60 

41 

0-75 

60 

26-00 

23 

10-90 

42 

58  THE      CARBOHYDRATES.  [CH.    II. 

112.     The  estimation  of  Cane-sugar. 

Boil  40  c.c.    of  the   solution  with  30  c.c.   of  —  hydrochloric 

acid  keeping  the  mixture  boiling  for  1  minute.     Cool,  neutralise  by 

N 
adding  30  c.c.  of  —  sodium  hydroxide,  cool  to  15°C.  and  make  the 

volume  up  to  100  c.c.  Estimate  the  amount  of  invert  sugar  in 
this  solution  by  either  of  the  methods  given  in  the  previous  exer- 
cises. 

Calculation  of  results. 

..      25  c.c.  Benedict's  solution  =  -0475  gm.  cane  sugar. 
10  c.c.  Fehling's         ,,         =  '0475  gm.     ,,         „ 

In  Bang's  method  calculate  as  glucose  and  multiply  by  0*95. 
Estimation  of  Maltose  and  Lactose. 

These  are  estimated  by  the  same  methods  as  glucose,  different 
factors  being  employed  for  the  calculation. 

10  c.c.  Fehling's  solution  |  =:  0*0676  gm.  lactose. 
25  c.c.  Benedict's       ,,       *  —  0*074    gm.  maltose. 


CHAPTER  III. 

THE  FATS  AND  THEIR  DECOMPOSITION 
PRODUCTS. 

The  fats  are  glycerine  esters  of  the  higher  fatty  acids. 

An  ester  is  a  compound  formed  by  the  condensation 
of  an  alcohol  with  an  acid. 

C2H5.OH  +  HOOC.CH,  C2H5.OOC.CH3  +  H2O 

Ethyl  alcohol.    Acetic  acid.     Ethyl  acetate. 

(Ethyl  ester  of  acetic  acid). 

Glycerine  is  a  trivalent  alcohol 
CH.OH 

or  C3H5(OH)3. 


It  can  therefore  condense  with  three  molecules  of  a  fatty 
acid. 

The  fatty  acids  found  combined  with  glycerine 
are  mostly  palmitic  acid  (C15H31.COOH),  stearic  acid 
(C17H,5.COOH)  and  oleic  acid  (C]7H33.COOH).  The  fats 
formed  by  the  condensation  of  glycerine  with  these 
acids  are  known  as  palmitin,  stearin  and  olein  (or  tri- 
palmitin,  etc.) 

CH2OH    HOOC.CWH81  CH2.OOC.C15H31 

CHOH  +  HOOC.C15H31  CH.OOC.C15H31  +  3H2O 


1531 


CH2OH    HOOC.C15H31  CH2.OOC.C15HH1 

or  C3H5(OOC.C15H31)3 
Palmitin. 


60  THE     FATS.  [CH.    III. 

Properties  of  tlie  fats. 

The  fats  are  solids  with  a  low  melting  point,  triolein 
melting  at  -5°C.,  tripalmitin  at  65° C.,  and  tristearin  at  71° C. 
In  the  body  they  are  found  mixed  in  different  proportions, 
and  the  melting  point  of  the  mixture  is  lower  the  greater 
the  percentage  of  triolein.  They  are  insoluble  in  water, 
salt  solutions  and  dilute  acids  and  alkalies.  They  are 
soluble  in  ether,  alcohol,  chloroform  and  a  variety  of 
organic  solvents. 

They  are  hydrolysed  by  boiling  acids  and  alkalies,  by 
superheated  steam  and  by  certain  enzymes,  called  lipases 
or  steapsins.  By  this  means  they  are  split  into  their 
constituents,  glycerine  and  fatty  acid.  If  an  alkali  is  used 
as  the  hydrolytic  reagent,  the  fatty  acid  combin'es  with  it 
to  form  a  soap.  This  special  form  of  hydrolysis  is  there- 
fore called  saponification. 

Various  methods  have  been  devised  for  the  identifica- 
tion of  the  fats,  amongst  them  being  : 

1.  The  melting  point. 

2.  The  saponification  figure.     A  known  weight  of  the 
fat  is  hydrolysed  by  means  of  a  known  amount  of  standard 
potash.     The   excess   of  alkali  is  then  titrated,  and  the 
number  of  decigrams  required  for  the  hydrolysis  of  100 
grams  of  the  fat  is  calculated. 

3.  The  iodine  number.     Oleic  acid  is  an  uiisaturated 
acid,  and  can  combine  with  two  atoms  of  iodine.     The 
amount  of  iodine  that  combines  with  100  grams  of  fat  can 
be  determined,  and  thus  the  percentage  of  uiisaturated 
acids  in  the  mixture  calculated. 

The   eniulsification  of  the  fats. 

Fats  can  be  emulsified,  i.e*  broken  up  into  droplets, 
either  mechanically  by  agitation,  or  "  spontaneously." 


CH.    III.]  DIGESTION    OF    FATS.  61 

The  mechanical  emulsificatioii  is  only  permanent  if 
the  droplets  are  surrounded  by  a  film  of  protein  (as  in 
milk),  or  by  a  film  of  soap  or  other  more  or  less  colloidal 
substance. 

"  Spontaneous "  emulsificatioii  takes  place  when  a 
melted  oil  or  fat  that  contains  a  certain  percentage  of  free 
fatty  acid  is  brought  into  contact  with  an  alkali.  The 
fatty  acid  dissolves  in  the  alkali  to  form  a  soluble  soap, 
and  the  diffusion  currents  thus  set  up  break  the  globule 
of  fat  into  small  particles,  the  process  being  maintained  by 
the  continual  exposure  of  fatty  acid  to  the  alkali.  The 
fat  in  the  small  intestine  is  thus  emulsified  as  a  prelimi- 
nary to  complete  hydrolysis  by  the  pancreatic  lipase. 

The  digestion  of  fats. 

The  fats  are  hydrolysed  to  a  small  extent  in  the 
stomach  by  gastric  lipase.  This  action  is  greater  if  the 
fat  be  given  in  an  emulsified  form,  as  in  milk. 

In  the  duodenum,  the  fat  mixed  with  the  fatty  acid  is 
spontaneously  emulsified  by  the  alkaline  bile,  succus 
entericus  and  pancreatic  juice.  The  emulsified  fat  is  then 
completely  hydrolysed  to  glycerine  and  fatty  acids  by  the 
pancreatic  lipase.  The  fatty  acids  are  converted  into  soluble 
soaps  by  the  alkalies. present.  The  soaps  and  glycerin  are 
absorbed  into  the  epithelial  cells  bordering  the  villi,  where 
they  are  resynthesised  into  fats.  These  are  passed  into 
the  lacteals  and  reach  the  blood  stream  by  way  of  the 
thoracic  duct. 

113.  (a)  Carefully  allow  a  drop  of  neutral  olive  oil  to  fall 
gently  on  to  the  surface  of  some  '25  per  cent.  Na2CO3  contained  in 
a  watch-glass.  The  drop  of  oil  remains  quite  clear  and  forms  a 
thin  circular  film  on  the  surface. 

(6)  Shake  5  c.c.  of  neutral  oil  with  3  drops  (only)  of  oleic  acid 
in  a  dry  test  tube.  With  a  drop  of  this  mixture  repeat  (a)  using  a 


62  THE    FATS.  [CH.    III. 

fresh  watch-glass  full  of  Na2CO3.  The  rancid  oil  slowly  spreads 
out  in  an  amoeboid  fashion  and  becomes  converted  into  a  milky 
emulsion. 

(c)  To  the  remainder  of  the  mixture  of  oil  and  oleic  acid  add 
12  more  drops  of  oleic  acid,  shake  well  and  repeat  the  experiment. 
The  drop  becomes  white  and  opaque,  but  does  not  become  emulsified. 

NOTES — 1.  It  is  absolutely  essential  that  the  oil  be  quite  neutral,  and  this 
can  best  be  tested  by  dropping  it  on  to  '25  per  cent.  Na2COs.  If  a  spontaneous 
emulsion  is  formed,  a  fresh  sample  must  be  obtained,  or  melted  fresh  butter 
substituted. 

2.  The  spontaneous  emulsion  in  (b)  is  caused  by  the  trace  of  oleic  acid 
dissolving   in    the  alkali  to  form  a  soap,  diffusion  currents  being  thus  set  up 
•which  divide  the  fat  into  microscopic  droplets. 

3.  In  (c)  the  large  excess  of  oleic  acid  leads  to  the  opaque  ring  of  soap 
being  formed  round  the  oil,  and  this  soap,  being  only  slightly  soluble  in  water, 
prevents  the  formation  of  an  emulsion. 

114.  Shake  a  few  drops  of  olive  oil  with  5  c.c.  of  ether  in  a 
dry  tube.     The  oil  completely  dissolves.     Repeat  the  experiment 
with  alcohol  instead  of  ether.     The  oil  dissolves  partially,  but  is  not 
so  soluble  in  alcohol  as  in  ether.     Pour  the  alcoholic  solution  into 
water.     The  fat  is  precipitated  as  an  emulsion. 

115.  Touch  a  piece  of  writing  paper  with  a  glass  rod  that  has 
been  dipped  in  olive  oil.     The  paper  is  rendered  translucent. 

Preparation  of  pancreatic  lipase. — A  perfectly  fresh  pig's  pancreas  is 
freed  from  fat,  weighed,  finely  minced  and  ground  with  sand.  It  is  then 
treated  with  three  times  its  weight  of  water  and  its  own  weight  of  strong  alcohol. 
It  is  allowed  to  stand  for  three  days  at  room  temperature  and  strained  through 
muslin.  It  must  not  be  filtered.  When  not  in  use  it  should  be  kept  in  a 
refrigerator.  It  will  remain  active  for  a  considerable  time. 

NOTE.  Pancreative  lipase  is  a  ferment  that  only  acts  with  the  co-operation 
of  a  co-ferment,  which  is  soluble  in  water  and  not  destroyed  by  boiling.  Bile 
salts  and  certain  other  substances  can  act  as  the  co-ferment.  The  ferment 
proper  is  practically  insoluble  in  water,  and  is  destroyed  by  boiling.  If  the 
pancreatic  extract  be  filtered,  neither  the  precipitate  nor  the  filtrate  has  any 
appreciable  action  on  fats ;  but  when  the  two  are  mixed  the  original 
lipolytic  action  is  recovered.  The  precipitate  is  the  ferment ;  the  filtrate 
contains  the  co-ferment. 

Preparation  of  an  Emulsion  of  Fat.— Commercial  olive  oil  (which 
contains  some  free  oleic  acid)  is  treated  in  a  flask  with  1  drop  of  a  1  per 


CH.    III.]  ACTION    OF    LIPASE.  63 

cent,  alcoholic  solution  of  phenolphthalein  for  every  10  c.c.  of  oil. 
Decinormal  sodium  hydroxide  is  added,  with  frequent  shaking,  till  the 
mixture  is  neutral.  A  very  stable  emulsion  is  thus  formed,  and  thus  a 
considerable  surface  of  fat  is  exposed  to  the  action  of  the  ferment. 

Fat-splitting  action  of  lipase  (steapsin). 

116.  Label  three  test  tubes  A,  B  and  C. 

To  A  add  2  c.c.  of  pancreatic  extract  and  1  c.c.  of  water. 
„  B     „    2  c.c.  ,,  „         boil,  and  add  1  c.c.  of  water. 

,,  C     ,,     2  c.c.  ,,  ,,         and  1  c.c.  of  1%  bile  salts. 

To  each  add  5  c.c.  of  the  emulsion  of  oil,  shake  thoroughly  and 
place  in  a  water  bath  at  40°  C.  for  1  hour. 

N 
Titrate  each  tube  with  —  NaOH  from  a  burette  and  note  the 

volume  required  to  make  the  solution  neutral.  The  amount 
required  for  B  is  a  measure  of  the  acidity  of  the  2  c.c.  of  pancreatic 
extract.  This  deducted  from  the  amount  required  for  A  and  C  is  a 
measure  of  the  amount  of  fatty  acid  formed  by  the  action  of  the 
ferment.  It  is  greater  in  C  than  A,  indicating  the  adjuvant  action 
of  bile  salts  on  the  lipolytic  action. 

117.  Shake  5  c.c.  of  neutral  olive  oil  in  a  test  tube  with  2  c.c. 
of  the  extract  of  the  pancreas  and  place  the  tube  in  a  water  bath  at 
37°  C.     At  the  end  of  every  ten  minutes  pipette  off  a  little  of  the  oil 
that  rises  to  the  surface,  allow  a  drop  of  it  to  fall  gently  on  to  some 
"25  per  cent.  Na2CO3  contained  in  a  watch-glass,  return  the  rest  to 
the  tube,  shake  vigorously  and  return  it  to  the  warm  bath.     As  the 
action  of  the  ferment  proceeds  spontaneous  emulsion  will   occur, 
showing  that  some  of  the  neutral  oil  has  been  converted  into  a  fatty 
acid.     If  the  action  is  allowed  to  proceed  considerably  further  no 
emulsion  will  be  produced,  for  the  reasons  stated  in  the  notes  to 
Ex.  113. 

NOTE. — This  is  one  of  the  methods  employed  for  demonstrating  the  fat- 
splitting  power  of  steapsin  ;  but,  naturally,  it  can  only  be  used  when  perfectly 
neutral  oil  can  be  obtained. 

118.  Repeat  the  above  experiment,  but  boil  and  then  cool  the 
2    c.c.    of    pancreatic    extract    before    adding   the    olive    oil.        A 


64  THE    FATS.  [CH.    III. 

spontaneous  emulsion  is  not  formed  at  any  stage,  showing  that  the 
ferment  is  destroyed. 

NOTES. — 1.  This  or  a  similar  control  experiment  should  always  be  per- 
formed side  by  side  with  the  actual  experiment  when  investigating  the  action  of 
ferments. 

2. — Be  particularly  careful  to  cool  the  extract  after  boiling,  otherwise  the 
alkali  may  exert  a  slight  saponifying  action  at  the  higher  temperature. 

119.  Boil  10  c.c.  of  fresh  milk,  cool  it  under  the  tap,  add  2  c.c. 
of  the  pancreatic  extract,  3  c.c.  of  litmus  solution  and  2  c.c.  of  2  per 
cent,  sodium  carbonate.      Shake  well  and  divide  into  two  portions, 
A  and  B.     Boil  A  to  destroy  the  ferment.     Place  both  tubes  in  the 
water  bath  at  37°  C.     In  the  course  of  ten  minutes  or  so  the  blue 
colour  in  tube    B   will  change  to  red,  indicating  that  some  of  the 
neutral  fat  in  the  milk  has  been  hydrolysed  to  a  fatty  acid. 

NOTE. — This  is  the  most  convenient  method  for  the  recognition  of  the 
action  of  lipase.  The  fat  of  milk  being  finely  emulsified  offers  a  very  large 
surface  for  the  action  of  the  steapsin.  The  milk  should  be  boiled  first  to  destroy 
any  bacilli  present  that  might  form  lactic  acid  from  the  lactose. 

Glycerine  (Glycerol). 

120.  Treat  a  drop  or  two  of  glycerine  in  a  test-tube  with  a 
solution  of  copper  sulphate  and  then  with  sodium  hydroxide.     A  blue 
solution  is  obtained,  glycerine  preventing  the  precipitation  of  cupric 
hydroxide. 

121.  Boil  the  solution  thus  obtained.      Reduction   does  not 
occur. 

122.  Heat  strongly  a  drop  or  two  of  pure  glycerine  with  solid 
potassium   hydrogen   sulphate    in   a   dry   test   tube.     The  pungent 
odour  of  acrolein  (acrylic  aldehyde)  is  noticed. 

CH2OH.CHOH.CH2OH  =  CH2 :  CH.CHO  +  2H2O 

Glycerine.  Acrolein. 

123.  Treat  about  5  c.c.  of  a  0'5  per  cent,  solution  of  borax 
with  sufficient  of  a  1  per  cent,  alcoholic  solution  of  phenolphthalein 
to  produce  a  well-marked  red  colour.     Add  a  20  per  cent,  aqueous 
solution   of  glycerine,  drop   by  drop,   until  the   red  colour  is  just 


CH.    III.]  HIGHER    FATTY    ACIDS.  65 

discharged.  Boil  the  solution :  the  colour  returns,  provided  that 
an  excess  of  glycerine  has  not  been  added  (Dunstan's  test  for 
glycerine). 

NOTES. — 1.     Any  ammonium  salt  will    discharge  the  colour,  but  in  this 
case  it  does  not  return  on  heating. 

2.  Any  polyhydric  alcohol  is  likely  to  give  the  same  reaction.     The  sugars 
are  all  polyhydric  alcohols,  but  are  distinguished  from  glycerine  by  their  reducing 
properties,   etc.,   and  by   the  fact  that  they  are  not  volatile  when  distilled  by 
steam. 

3.  ,The  probable  explanation  of  the  reaction  is  as  follows.     Sodium  borate 
is  partially  hydrolysed  in  aqueous  solution  to  boric  acid  and  sodium  hydroxide. 
Boric  acid  being  a  weak   acid  is  only  feebly  ionised  and  therefore  the  solution 
reacts   alkaline.     On  adding  glycerine,  glyceroboric  acid  is  formed.      This  is  a 
strong  acid  and  hence  the  reaction  of  the  solution  changes  from  alkaline  to  acid. 
On  heating,    unless  a  large  excess  of  glycerine  be  present,  the  glyceroboric  acid 
is  hydrolysed  to  glycerine  and  boric  acid  and  the  solution  again  becomes  alkaline. 

The  Higher  fatty  acids  and  their  salts,  the  soaps. 

124.  Shake    a  few  drops  of  oleic  acid  with  5  c.c.   of  water, 
ether,    and   alcohol    respectively    in    separate  tubes.     The   acid    is 
insoluble  in  water,  but  soluble  in  alcohol  or  -ether. 

125.  Place  a  drop  of  oleic  acid  on  writing  paper  :  a  greasy 
stain  results. 

126.  Shake  the  alcoholic  solution   of  oleic  acid  with   dilate 
bromine  water.     The  colour  of  the  bromine  is  discharged,  owing  to 
the  unsaturated  acid  absorbing  the  halogen  till  it  is  saturated. 

127.  Repeat    the   experiment   with   an   alcoholic   solution  of 
stearic  acid  or  commercial  "  stearine "   (a  mixture  of  stearic  and 
palmitic   acids).     The    colour  of  the  bromine  persists,  since  these 
acids  are  members  of  the  saturated  series. 

128.  Heat  about  10  drops  of  oleic  acid  with  10  c.c.  of  water 
and  to  the  hot  mixture  add  40  per  cent.  NaOH  drop  by  drop  till 
the  solution  is  clear.     If  an  excess  be  added  the  excess  of  sodium 
ions   causes    a  precipitate    (see   note   below).     A   clear  solution  of 
a  soap,  sodium  oleate,  is  formed.     Divide  this  into  three  portions. 

F 


66  THE    FATS.  [CH.    III. 

To  A  add  a  few  drops  of  strong  HC1  or  H2SO4  till  the  reaction 
is  distinctly  acid.  Oleic  acid  separates  out  and  rises  to  the  surface 
of  the  tube. 

To  B  add  finely-powdered  sodium  chloride  and  shake.  The 
soap  is  rendered  insoluble  and  rises  to  the  surface. 

To  C  add  some  calcium  chloride.  A  precipitate  of  an 
insoluble  soap,  calcium  oleate,  is  produced. 

NOTE. — B  illustrates  the  principle  of  "salting  out,"  which  is  used  in 
the  manufacture  of  soaps.  The  excess  of  sodium  ions  in  the  solution,  produced 
by  the  addition  of  the  sodium  chloride,  lowers  the  solubility  of  the  sodium 
oleate,  which  is  therefore  precipitated. 

129.  Boil  2  c.c.  of  olive  oil  with  5  c.c.  of  a  20  per  cent, 
alcoholic  solution  of  sodium  hydroxide  in  a  basin  over  a  small  flame 
for  five  minutes  or  until  the  alcohol  has  all  evaporated  away.  Add 
about  5  c.c.  of  alcohol  and  heat  again  to  dryness,  stirring  the  whole 
time.  Add  about  30  c.c.  of  distilled  water  and  boil  till  dissolved. 
Add  solid  sodium  chloride  and  stir.  The  soap  formed  is  precipitated. 
Filter  some  off,  dissolve  in  boiling  water  and  repeat  the  experiments 
described  in  the  previous  exercise. 


CHAPTER    IV. 
THE    CHEMISTRY    OF    SOME    FOODS. 

A.    Milk. 

The  composition  of  milk  differs  considerably  in 
different  animals. 

The  percentage  composition  of  average  samples  of 
human  and  cow's  milk  is  as  follows  :— 

Carbo- 
Protein.      Fat.     hydrate.      Salts. 

Human 1-5  3-1  5'0  O2 

Cow's 3-4  3-7  4-8  0'7 

Other  differences  are  that  in  cow's  milk  the  propor- 
tion of  caseinogen  to  lactalbumin  is  about  6  to  1  compared 
with  2  to  1  in  human  milk. 

Caseinogen,  the  chief  protein  of  milk,  is  a  phospho- 
protein.  It  is  insoluble  in  water,  dilute  acids  and  salts, 
but  dissolves  in  alkalies  to  form  a  salt-like  body.  It  also 
dissolves  in  strong  acids.  It  is  salted  out  of  solution 
by  half -saturation  with  ammonium  sulphate. 

It  does  not  coagulate  on  boiling.  But  when  milk  is 
boiled  a  skin  forms  on  the  surface.  A  similar  skin  forms 
whenever  a  protein  solution  mixed  with  an  emulsion  of  a 
fat  is  heated.  The  skin  contains  protein  mixed  with  fat. 
If  it  be  removed,  another  skin  immediately  forms. 

130.  Examine  a  drop  of  fresh  cow's  milk  under  the  micro- 
scope with  a  high  power.  Notice  the  highly-refractive  fat  globules 
of  varying  size,  the  smallest  globules  exhibiting  the  peculiar  vibra- 
tion known  as  Brownian  movement. 


68  THE     CHEMISTRY     OF     SOME     FOODS.  [cH.    IV. 

131.  Take  the  specific  gravity  of  milk  with  a  lactometer.     It 
varies  between  1028  and  1034. 

NOTE.  —When  the  milk  is  skimmed  the  specific  gravity  rises  from  1033  to 
1037,  owing  to  the  removal  of  the  fat  which  has  a  low  specific  gravity.  The 
specific  gravity  is  also  lowered  by  dilution  with  water. 

132.  Place  a  drop  of  fresh  milk  on  pieces  of   blue  and  red 
litmus  paper  and  wash  off  with  distilled  water.     The  blue  paper  is 
turned  red  and  the   red  paper  blue,  i.e.  the  milk  is  amphoteric   in 
reaction,  due  to  the  mixture  of  acid  and  alkaline  salts. 

133.  Take  5  c.c.  of  milk  in  a  test  tube  and  dilute  with  dis- 
tilled water  till  the  test  tube  is  nearly  full.     Add   three   drops   of 
strong  acetic  acid  and  mix  thoroughly.     A  flocculent  precipitate  of 
caseinogen  is  formed,  which  mechanically  carries  the  fat  down  with 
it.     Filter  this  off  and  label  the  filtrate  A.     Precipitate  two   more 
portions  of  5  c.c.  each,  adding  the  filtrates  to  A,  and  reserving  the 
precipitate. 

134.  Take  5  c.c.  of  milk,  add  water  as  before,  and  then  an 
excess  of  strong  acetic  acid.     A  precipitate  is  not  produced,  owing 
to  the  solubility  of  caseinogen  in  an  excess  of  acid. 

135.  Treat  a  portion  of  the  precipitate   from   Ex.    133  with 
some    2    per    cent.    Na2CO3    solution.     The    caseinogen    dissolves, 
leaving  the  fat  in  suspension.     Apply  the  protein  colour  reactions 
to  the  solution :  all,  except  the  sulphur  test,  are  given. 

136.  Treat  5  c.c.  of  milk  with  5  c.c.  of  saturated  ammonium 
sulphate   solution.     The  caseinogen  is  precipitated,  entangling  the 
fat    with    it.     Filter   and    boil  the  filtrate.     A    heat    coagulum  of 
lactalbumin  is  obtained.     Treat  the  precipitate  of  caseinogen  and 
fat  on  the  paper  with  water.     The  caseinogen  dissolves. 

NOTE. — The  caseinogen  dissolves  in  water  because  it  is  precipitated  as  a 
salt  by  ammonium  sulphate.  On  the  addition  of  dilute  acetic  acid  to  this 
solution,  a  precipitate  of  caseinogen  is  again  obtained. 

137.  Treat  a  considerable  portion  of  the  precipitate  obtained 
in  Ex.   133  as   directed  in   Ex.   39.       Phosphorus   is  found  to  be 
present  in  the  caseinogen. 


CH.    IV.]  MILK.  69 

138.  Allow  another   portion    of    the    precipitate    obtained    in 
Ex.   133  to  drain   thoroughly,   press   it  with  dry   filter  paper  and 
transfer  it  to  a  dry  tube.     Shake  it  vigorously  with  5  c.c.  of  ether, 
pipette  off  the  ether,  and  evaporate  the  etheral  solution  in  a  basin 
over  a  boiling  water  bath,  turning  out  the  flame  before  putting  on 
the  dish  containing  the  ether.     A  small  amount  of  fat  is  left  in  the 
dish.     Wipe  the  dish  round  with  a  piece  of  writing-paper.     A  trans- 
lucent grease  spot  is  formed. 

139.  Examine  filtrate  A.     Add  a  drop   of  litmus,   and  note 
that  it  is  markedly  acid. '    Boil,  and  whilst  boiling  add  2  per  cent. 
Na2CO3,  drop  by  drop,  until  the  reaction  is  only  faintly  acid.     If 
the  reaction   should,  by  accident,  be  made   alkaline,   dilute  acetic 
acid  must  be  added  till  the  reaction  is  faintly  acid.     A  coagulum  of 
lactalbumin  is  formed.     Filter  this  off  and  reserve  the  filtrate  (B). 

140.  Boil  a  small  portion  of  filtrate  B  with  a  little  Fehling's 
solution.     A  well-marked  reduction  is  obtained,  due  to  the  presence 
of  lactose. 

141.  Try  Barfoed's  reaction  with  this  filtrate.     A  reduction  is 
not   usually  obtained.      (See   Ex.  69.)      Sometimes  the   lactose   is 
slightly  hydrolysed  by  the  boiling  in  Ex.   139. 

142.  Treat  the  remainder  of  filtrate  B  with  two  or  three  drops 
of  strong  ammonia  and  boil.     A  slight  precipitate  of  calcium  phos- 
phate  is  produced.     Filter  this   off,   dissolve   it   in  a  little    strong 
acetic   acid,   and  add   a   solution   of   potassium  oxalate.      A  white 
precipitate  of  calcium  oxalate  is  formed.      .Treat  with  2  c.c.  of  nitric 
acid  and  5  c.c.  of  ammonium  molybdate  solution.     Boil  for  two 
minutes.     A  yellow  crystalline  precipitate  is  formed,  showing  the 
presence  of  phosphates  in  milk. 

B.    The  Clotting  of  Milk. 

When  milk  is  treated  with  a  neutral  or  faintly  acid 
extract  of  the  mucous  membrane  of  the  stomach,  a  clot 
forms  after  a  certain  time.  This  is  due  to  the  conversion 
of  the  caseinogen  of  the  milk  into  an  insoluble  protein 


70  THE     CHEMISTRY     OF     SOME     FOODS.  [CH.    IV. 

called  casein.  This  entangles  the  greater  portion  of  the 
fat,  the  whole  being  known  as  the  curd.  The  fluid 
portion  that  separates  from  the  curd  is  called  the  whey, 
and  contains  the  salts,  lactose  and  lactalbumin. 

The  conversion  of  caseinogen  into  casein  was  believed 
at  one  time  to  be  due  to  a  special  enzyme  called  rennet  or 
rennin.  But  it  is  probable  that  the  action  is  one  common 
to  all  proteolytic  enzymes,  as  trypsin  and  erepsin  can 
cause  milk  to  clot. 

Soluble  ionised  calcium  salts  participate  in  the 
clotting  action,  their  role  being  to  convert  a  soluble 
product  of  ferment  action  into  an  insoluble  one.  The 
mechanism  of  clothing  is  shewn  in  the  following  scheme : — 

Caseinogen.     Proteolytic  enzyme.     Soluble  calcium  salt. 

(Rennet) 


Soluble  protein  +  Soluble  casein 

resembling  albumose. 


Insoluble  casein. 

The  following  experiments  can  be  performed  with  a  commercial 
preparation  of  rennet: 

143.  Treat    5  c.c.    of .» milk   with   about    2  c.c.    of   an    active 
solution  of  rennet-ferment.     Place  the  tube  in  the  warm  bath,  and 
observe  it  from  time  to  time.     Note  that  the  milk  soon  forms  a  clot 
so  firm  that  the  tube  can  safely  be  inverted  :  on  standing  longer  the 
clot  contracts  and  exudes  a  nearly  clear  fluid  (whey). 

144.  Perform  a  control  test  by  boiling  and  then  cooling  the 
rennet  before  adding  it  to  the  milk.     Clotting  does  not  take  place. 

14j.  Treat  5  c.c.  of  milk  with  2  c.c.  of  2  per  cent.  Na2CO3 
and  the  same  amount  of  rennet :  place  the  tube  in  the  warm  bath. 
Clotting  does  not  take  place. 


CH.    IV.J  CLOTTING    OF    MILK.  71 

NOTE. — Commercial  rennin  is  prepared  from  the  fourth  stomach  of  a 
sucking  calf,  or  from  the  mucous  membrane  of  the  stomach  of  a  pig.  The 
pepsin,  and  so  also  the  rennetic  action,  is  destroyed  by  alkalies. 

146.  Take    lOc.c.   of   milk,   add   one-third  of   its  volume   of 
1  per  cent,  potassium  oxalate  (to  remove  all  soluble  calcium  salts) 
and    divide    into  three   equal   portions   which  are   placed  in  three 
test-tubes,  labelled  A,   B  and  C. 

To  A  add  1  c.c.  of  2  per  cent,  calcium  chloride  and  2  c.c.  of 
rennet. 

To  B  add  2  c.c.  of  rennet. 

To  C  add  2  c.c.  of  boiled  rennet.  x 

Place  the  three  tubes  in  the  warm  bath  for  about  ten  minutes. 
Note  that  A  clots  and  that  B  and  C  do  not. 

Boil  B  (to  destroy  the  rennet)  and  cool  the  tube. 

To  B  and  C  add  1  c.c.  of  2  per  cent,  calcium  chloride. 

A  flocculent  precipitate  of  insoluble  casein  is  immediately 
formed  in  B  :  in  C  there  is  no  precipitate. 

NOTE. — In  A  there  is  caseinogen,  rennet  and  CaCls  • 

In  B  ,,         ,,  and  rennet. 

In  C  „         „  and  CaCl2 

After  ten  minutes,  B  contains  soluble  casein,  which  is  precipitated  by 
the  subsequent  addition  of  CaCl2. 

G.    Cheese. 

147.  Shake  some  grated  cheese  in  a  dry  test  tube  with  ether, 
and  examine  the  ethereal  solution  for  fat  as  in  Exercise  138.     Fat 
is  present  in  considerable  quantity. 

148.  Pound    the    residue    from    the  above  in  a  mortar  with 
a   2   per   cent,   solution   of  sodium  carbonate  and  filter.      Acidify 
a  portion  of  the  filtrate.     A  precipitate  of  casein  is  formed,  which  is 
soluble  in  excess  of  acid.     To  the  remainder  of  the  filtrate  apply 
the  usual  protein  colour  reactions :  they  are  all  obtained. 

D.    Potatoes. 

149.  Scrape  the  clean  surface  of  half  a  potato  with  a  pen- 
knife,   keeping   the    scrapings    as    fine    as    possible.      Place    the 


72  THE    CHEMISTRY    OF    SOME     FOODS.  ^CH.    IV. 

scrapings  in  a  beaker  of  water,  stir  well,  and  strain  through  fine 
muslin  into  another  beaker.  Allow  this  to  stand  for  a  few  minutes 
and  then  note  the  white  deposit  of  starch.  Pour  off  the  supernatant 
fluid  and  reserve  it  for  the  next  exercise.  Fill  the  beaker  con- 
taining the  starch  with  water,  stir  well,  and  again  allow  the  starch 
to  settle.  By  repeating  this  process  of  lixiviation  the  starch  can 
be  obtained  quite  pure.  Examine  a  little  microscopically  and  note 
the  characteristic  form  of  the  grains  (See  Ex.  85).  Heat  a  little 
with  water,  cool,  and  add  iodine.  A  deep  blue  colour  is  obtained. 

150.  Filter  the  fluid  A,  and  test  portions  of  the  filtrate  for 
proteins  by  the  usual  colour  tests.     Only  small  quantities  of  protein 
are  found  to  be  present,  the  most  marked  reaction  being  Millon's. 

E.    Flour. 

White  flour  from  the  endosperm  of  wheat  grains 
contains  70  to  75  per  cent,  of  starch,  about  8  per  cent,  of 
protein  and  about  1  per  cent,  of  fat.  The  proteins  are 
gliadin  (soluble  in  70  to  80  per  cent,  alcohol),  and  glutelin 
(soluble  in  alkali).  When  treated  with  water  these  two 
proteins  form  a  sticky  mass  called  gluten,  the  viscidity 
being  due  to  the  gliadin.  Thus  grains  poor  in  gliadin,  as 
rice  and  oats,  do  not  form  dough  when  mixed  with  water. 

Flour  only  contains  glucose  if  germination  has  taken 
place  before  milling. 

Whole  flour  is  obtained  from  the  whole  of  the  grain, 
except  the  outer  husk  and  outer  part  of  the  bran.  It  is 
possible  that  it  contains  something  essential  to  growth 
and  general  nourishment.  It  is  not  quite  so  digestible  as 
white  flour.  The  bran  in  it  stimulates  the  intestine  and 
so  acts  as  a  mild  laxative. 

151.  Mix  some  wheat  flour  with  a  little  water  to  form  a  stiff 
dough.     Allow  this  to  stand  for  a  short  while,  preferably  at  37°  C. 

Wrap  a  piece,  the  size  of  a  chestnut,  in  muslin,  and  knead  it 
for  a  few  minutes  in  a  basin  of  water ;  pour  the  suspension  into  a 


CH.    IV.]  BREAD.  73 

beaker,  and  note  the  white  deposit  of  starch  grains  that  settles  down 
on  standing.  Examine  this  microscopically,  noting  that  the  grains 
differ  considerably  from  those  of  potato-starch  in  being  smaller, 
circular,  and  with  a  central  hilum.  Make  a  drawing  of  the  grains. 
Boil  a  little  with  water,  cool,  and  add  a  drop  of  iodine.  The  deep 
blue  starch  reaction  is  obtained. 

152.  Knead  the  dough  thoroughly  under  the  tap  until  no  more 
starch  comes  through  the  muslin.  A  yellowish,  sticky  mass,  known 
as  gluten,  is  left  behind.  Test  portions  of  this  by  the  usual  protein 
colour  reactions  :  they  are  all  obtained,  gluten  being  a  protein. 

F.     Bread. 

The  dough  formed  by  adding  water  to  flour  is  imper- 
vious to  the  digestive  juices.  Before  it  can  be  used  it  has 
to  be  aerated  and  the  gluten  rendered  porous. 

A  pure  culture  of  yeast  is  mixed  with  warm  water, 
flour  and  salt.  The  dough  thus  formed  is  thoroughly 
kneaded,  and  the  mass  kept  warm  for  some  hours. 
During  this  time  the  yeast  cells  multiply  and  convert 
some  of  the  starch  into  glucose  and  this  into  alcohol  and 
CO2.  Also  the  ferment  of  the  flour  called  diastase  becomes 
active  and  converts  some  of  the  starch  into  glucose.  More 
flour  is  added  and  the  process  allowed  to  proceed  for 
some  hours  longer.  The  gas  formed  causes  the  mass  to 
rise.  The  dough  is  weighed  out  into  loaves,  which  after 
being  allowed  to  rise  once  more  for  a  certain  time  are 
heated  to  about  232°  C.  for  an  hour  and  a  half.  The  heat 
kills  the  yeast,  expands  the  gas  bubbles,  and  causes  the 
outer  part  of  the  dough  to  become  hardened  by  coagulating 
the  proteins.  It  also  converts  starch  into  soluble  starch 
and  dextrin,  thus  forming  the  crust.  The  brown  appear- 
ance of  this  is  due  to  the  conversion  of  glucose  into 
caramel. 


74  THE    CHEMISTRY     OF     SOME     FOODS.  [CH.    IV. 

153.  Take  a  piece  of  the  crumb  of  a  stale  white  loaf,  rub  it 
up  finely  and  pound  with  cold  water  in  a  mortar.  Strain  and 
squeeze  through  muslin.  A  white  fluid  is  obtained  containing 
wheat  starch  grains.  Filter  the  fluid.  To  a  portion  of  the  filtrate 
add  a  little  Fehling's  solution  and  boil:  a  well  marked  reduction 
occurs  due  to  the  presence  of  glucose.  To  another  portion  add 
iodine :  a  purple  colour  is  produced,  showing  the  presence  of 
erythro-dextrin.  If  very  dilute  iodine  be  cautiously  added,  a  blue 
colour  is  produced  at  first,  showing  that  a  small  amount  of  soluble 
starch  is  present. 

Boil  a  small  amount  of  the  residue  of  the  bread  with  water  in  a 
beaker,  strain  through  muslin  and  filter.     Cool  and  test  the  filtrat 
for  starch  and  dextrin.     (Ex.  96  and  97.) 

154.  Repeat  the  above  exercise,  using  the  crust  of  bread 
instead  of  the  crumb.  Note  that  glucose  is  absent  or  present  in 
traces  only  :  dextrin  and  starch  are  present,  a  considerable  portion 
of  the  latter  existing  as  soluble  starch  and  being  present  in  the  cold 
water  extract. 

G.    Meat  (Muscle). 

The  most  important  constituents  of  living  striated 
muscle  are- 
Proteins.    Myosinogen  and  Paramyosinogen. 
Pigment.    Myohaematin. 
Fat. 

Nitrogenous  extractives.    Creatine. 

Hypoxanthine. 
Xanthine. 

Non-nitrogenous  extractives.     Glycogen. 

Sarcolactic  acid. 

Inorganic.    Water. 

Salts,  chiefly  potassium  and  magnesium 
phosphates. 


CH.    IV.]  MUSCLE.  75 

The  proteins  of  living  muscle  are  mainly  myosinogen 
(80  per  cent.)  and  paramyosinogen  (20  per  cent.).  The 
former  is  an  albumin,  coagulating  at  57°  C.  The  latter  is  a 
globulin,  coagulating  at  47°  C. 

On  standing  or  on  treatment  with  dilute  acids  they 
are  converted  into  myosin  the  protein  of  dead  muscle.  In 
this  transformation,  myosinogen  passes  through  an  inter- 
mediate stage  of  soluble  myosin  which  coagulates  at  40°  C. 

Myosinogen.  Paramyosinogen. 

Soluble  myosin. 

Myosin. 

155.  Preparation  of  fresh  muscle  extract.     A  rabbit  is 
killed,  a  cannula  fixed  into  the  aorta  and  an  opening  made  in  the 
right  auricle.     The  vessels  are  then  washed  free  from  blood  with 
0'9   per   cent,    sodium    chloride.      The  muscles   of    the  limbs  are 
removed,    rapidly   minced    and    treated  with    ice-cold   5   per  cent, 
magnesium  sulphate,  and  the  mixture  left  in  the  ice  chest  for  about 
24  hours.     The  extract  is  filtered  and  the  following  tests  performed 
with  it : 

156.  Take  the  reaction  to  litmus.     It  is  generally  neutral. 

157.  Dilute  a  small   portion  with   four  volumes  of  distilled 
water  and  leave  the  tube  in  the  water  bath  at  37°  C.  for  some  time. 
A  clot  of  myosin  forms,  leaving  muscle  serum. 

158.  Take  the  reaction  of  the  muscle  serum  to  litmus.     It  is 
distinctly  acid,  due  to  the  production  of  sarcolactic  acid. 

159.  Add  some  acetic  acid  to  another  portion  of  the  extract. 
A  precipitate  of  myosin  occurs  immediately. 

160.  Take  5  c.c.  of  the  extract  in  a  test-tube  :  place  the  tube 
in  a  beaker  of  water,  supporting  it  by  a  clamp  so  that  it  does  not 
touch  the  bottom  of  the  beaker.     Heat  the  water  with  a  Bunsen 
flame    and   note  the   temperature    in    the   tube   at    which    distinct 


76  THE    CHEMISTRY     OF     SOME     FOODS.  [CH.    IV. 

coagulation  occurs.  It  is  usually  at  about  47°  C.  Filter  off  the 
coagulum  of  paramyosinogen  and  heat  again.  Another  and  larger 
coagulum  of  myosinogen  occurs  at  57°  C. 

161.  Preparation  of  Myosin.     Fresh  veal  is  finely  minced 
in  a  machine,  stirred  with  a  large  volume  of  water  for  a  quarter  of 
an  hour,  strained  through  muslin,  and  the  washing  process  repeated 
once   more.      In  this   way  certain   proteins  and   other  substances 
soluble  in  water  are  removed.    The  veal  is  now  collected  on  muslin, 
squeezed  to  remove  the  water,   ground  with   sand,   and  extracted 
with  five  times  its  volume  of  10  per  cent,  ammonium  chloride  for 
several  hours  at  room  temperature.     The  extract  is  filtered  through 
muslin,  linen,  and  then  coarse  filter  paper.     In  this  way  a  crude, 
viscid  solution  of  myosin  is  obtained. 

162.  Boil  a  portion  of  the  solution.      A  heavy  coagulum  is 
formed.     Wash  the  coagulum  and  on  it  perform  the  protein  colour 
reactions.     They  are  all  obtained. 

163.  Pour   100  c.c.  into  a  litre  of  water  contained  in  a  tall 
cylinder ;  mix  well,  and  note  the  precipitation  of  myosin,  due  to  the 
reduction  in  the  concentration  of  salts. 

Allow  this  to  settle  and  then  pour  or  pipette  off  as  much  of  the 
supernatant  fluid  as  possible.  A  suspension  of  myosin  in  dilute 
ammonium  chloride  is  thus  obtained  for  the  next  three  experiments. 

NOTE. — If  this  suspension  be  allowed  to  stand  it  slowly  becomes  converted 
into  an  insoluble  variety. 

164.  To  a  portion  add  a  saturated  solution  of  common  salt, 
drop    by    drop.      The    precipitate    dissolves.      Add  solid   NaCl  to 
saturation  :  the  myosin  is  reprecipitated. 

165.  To  a  portion  add  saturated  (NH4)2SO4  till  the  precipitate 
just  dissolves.     Now  add  an  equal  bulk  of  saturated  (NH4)2SO4. 
The  myosin  is  reprecipitated. 

166.  Dissolve  in  a  little  (NH4)2SO4  and  take  the  temperature 
at  which  the   myosin   coagulates.       It  coagulates   at  about   57°  C. 
(See  Ex.  160.) 


CH.    IV.]  CREATINE.  77 

Creatine. — This  is  the  most  abundant  nitrogenous 
extractive  in  muscle,  being  present  to  the  extent  of  about 
0-4  per  cent.  Chemically  it  is  methyl-guanidine-acetic 
acid. 

NH  CH;! 

NH2  —  C  —  N  —  CH2  —  COOH. 

On  hydrolysis  with  baryta  water  it  is  converted  into 
urea  and  sarcosine  (methyl  glycine). 

NHCH3  NH2  CH3 


,00  + 


NH2.C  -  N.CH2.COOH  +  HaO  =  NH2.CO  +  NH.CH2.COOH. 

Urea.          Sarcosine. 

On  being  boiled  with  mineral  acids  it  is  dehydrated 
to  creatinine. 

NH  CH, 

||        I 
C  — 

NH  — CO. 

Creatinine  is  found  in  normal  human  urine,  but 
creatine  only  under  abnormal  conditions. 

167.  Separation  of  creatine  from  mea^t  extract.  Dissolve 
10  grams  of  commercial  meat  extract  in  200  c.c.  of  water.  Add 
slowly  a  saturated  solution  of  lead  acetate  till  no  further  precipitate 
is  formed,  carefully  avoiding  an  excess.  This  is  best  done  by 
filtering  samples  and  testing  them  with  lead  acetate.  Filter  off  the 
precipitate  of  proteins  and  phosphates.  Warm  the  filtrate  and 
decompose  the  soluble  lead  compounds  by  means  of  a  stream  of 
sulphuretted  hydrogen.  Warm  and  filter  off  the  precipitate  of 
lead  sulphide.  Evaporate  the  filtrate,  filtering  off  any  sulphur  or 
sulphide  that  may  be  deposited.  Continue  the  evaporation  till  a 
syrup  is  obtained.  Allow  this  to  stand  in  the  ice  chest  for  two  or 
three  days.  Creatine  separates  out,  mostly  as  oblique  rhombic 
crystals.  Examine  a  few  under  the  microscope.  Treat  the  syrup 


78  THE    CHEMISTRY    OF    SOME     FOODS.  [~CH.    IV. 

with  200  c.c.  of  88  per  cent,  alcohol,  stir  thoroughly  with  a  glass 
rod  and  filter  through  a  small  paper.  The  creatine  remains  on  the 
paper,  the  alcoholic  filtrate  containing  the  purine  bases. 

168.  Conversion  of  creatine  into  creatinine.     Dissolve  the 
creatine  in  about  30  c.c.  of  hot  water  and  divide  the  solution  into  two 
equal  portions,  A  and  B.     Treat  B  with  an  equal  volume  of  normal 
HC1  and  heat  on  a  boiling  water  bath  in  a  flask  fitted  with  a  cork 
and  long  glass  tube  (to  act  as  an  air  condenser)  for  three  to  five 
hours.     The  creatine  is  converted  into  creatinine.     Neutralise  the 
solution  with  caustic  soda. 

Test  A  and  B  for  creatinine  by  the  following  tests : 

169.  Jaffe's  test  for  creatinine.     Treat  10  c.c.  of  the  solution 
with  15  c.c.  of  saturated  picric\acid  solution  and  5  c.c.  of  10  per 
cent,  caustic  soda.     Allow  the  mixture  to  stand  for  5  minutes  and 
dilute  to  200  c.c.     A  deep  orange  colour  appears  in  B  due  to  the 
formation  of  picramic  acid  from  creatinine.     The  creatine  in  A  gives 
no  colour. 

170.  Weyl's  test  for  creatinine.    Treat  5  c.c.  with  a  few 
drops  of  a  freshly  prepared  solution  of  sodium  nitroprusside  and 
make  the  solution    alkaline  with  sodium  hydroxide.       A  ruby-red 
colour  appears,  whicfh  soon  turns  yellow.     Acidify  with  an  excess 
of  acetic  acid  and  heat.     A  green  tint  appears,  and  a  blue  deposit 
of  Prussian  blue  may  result  on  standing. 

Purine  bases.  These  compounds  are  interesting 
because  of  their  chemical  relationship  to  uric  acid.  This 
relationship  is  shown  by  the  formulae  given  on  page  20. 

The  purine  bases  found  in  meat  extracts  are  chiefly 
hypoxanthine  and  xanthine.  They  can  be  obtained  from 
the  alcoholic  solution  obtained  in  Ex.  166,  by  evaporating 
off  the  alcohol,  adding  ammonia  and  precipitating  with 
ammoniacal  silver  nitrate. 


CH.    IV.]  SARCOLACTIC    ACID.  79 

Sarcolactic  acid  is  dextro-rotatory  a-oxy-propionic  acid. 

CHS  CH3 

I  I 

H-C-OH       or       HO-C-H 

COOH  COOH 

The  middle  carbon  atom  of  this  compound  is  attached 
to  four  different  groups,  -  CH3,  -  H,  -  OH  and  -  COOH. 
Solutions  of  such  asymmetric  compounds  have  the  power 
of  rotating  the  plane  of  polarised  light,  either  to  the  right 
or  to  the  left. 

If  the  carbon  atom  be  represented  as  a  regular  tetra- 
hedron, and  the  four  different  groups  placed  at  the  apices, 
then  any  arrangement  of  the  groups  round  the  tetrahe- 
dron will  show  a  figure  which  is  reversed  by  its  image  in 
a  mirror.  Projected  on  to  a  plane  surface  the  above 
formulae  are  obtained.  The  first  of  these  is  dextro- 
rotatory, and  the  other  is  laevo-rotatory. 

If  an  asymmetric  compound  be  prepared  by  artificial 
synthesis,  it  consists  of  equal  amounts  of  d-  and  Z-forms, 
and  is  therefore  optically  inactive  (racemic  or  dl-). 

The  lactic  acid  found  in  muscle  is  d-lactic.  That 
formed  by  the  fermentation  of  lactose  and  other  carbo- 
hydrates is  generally  <iZ-lactic.  Certain  bacteria,  how- 
ever produce  Z-lactic  acid. 

Sarcolactic  acid  is  present  to  a  very  small  extent  in 
fresh  living  muscle.  The  amount  increases  rapidly  in 
fatigue,  especially  in  the  absence  of  a  proper  supply  of 
oxygen.  On  leaving  a  fatigued  muscle  in  an  atmosphere 
of  oxygen,  the  amount  of  lactic  acid  decreases. 

There  is  a  marked  production  of  lactic  acid  at  the 
onset  of  rigor  mortis.  But  if  a  fresh  muscle  be  suddenly 
coagulated  by  dropping  it  into  boiling  water,  there  is  no 
such  marked  production  of  the  acid. 


80  THE     CHEMISTRY    OF     SOME     FOODS.  [CH.    IV. 

It  is  probable  that  the  lactic  acid  appearing  in  fatigue 
and  in  rigor  arises  through  the  decomposition  of  some 
complex  material  in  the  muscle,  but  this  has  not  been 
definitely  established. 

Sarcolactic  acid  is  a  liquid,  soluble  in  water,  alcohol 
and  ether.  It  forms  a  characteristic  zinc  salt,  which  is 
obtained  by  boiling  a  solution  with  excess  of  zinc  carbo- 
nate, filtering  and  evaporating  slowly.  The  crystals 
contain  two  molecules  of  water  of  crystallisation,  the  zinc 
salt  of  ordinary  fermentation  lactic  acid  containing  three. 

171.  Hopkins'  reaction  for  lactic  acid.     To  3   drops  of 
a  1  per  cent,  alcoholic  solution  of  lactic  acid  in   a  clean,  dry  test 
tube  add  5   c.c.  of  concentrated  sulphuric  acid  and, 3  drops   of  a 
saturated  solution  of  copper  sulphate.     Mix  and  place  the  tube  in  a 
beaker  of   boiling  water  for  about  five  minutes.     Cool  thoroughly 
under  the  tap,  add  two  drops  of  a  '2  per  cent,  alcoholic   solution  of 
thiophene,  and  shake.     Replace  the  tube  in  the  boiling  water  bath. 
As  the  mixture  gets  warm  a  fine  cherry-red  colour  develops. 

NOTE. — Lactic  acid  is  oxidised  in  sulphuric  acid  solution  to  some  substance 
which  gives  a  red  colour  with  thiophene.  The  copper  sulphate  aids  this 
oxidation,  which  is  inhibited  by  water. 

172.  Uffelmann's  reaction  for  lactic  acid.    Treat  a  few  c.c. 
of  Uffelmann's  reagent  with  a  few  c.c.  of  a  dilute   (0'4  per  cent.) 
solution  of  lactic   acid.     The   violet  colour   is   instantly  turned  to 
a  yellow. 

NOTES. — 1.  Uffelmann's  reagent  is  prepared  by  treating  a  1  per  cent, 
solution  of  phenol  (carbolic  acid)  with  very  dilute  ferric  chloride  till  the 
solution  becomes  coloured  an  amethyst-violet. 

2.  The  reaction  is  not  very  reliable,  since  other  acids  as  tartaric,  oxalic 
and  citric  give  it. 

173.  The  Formation  of  Lactic  Acid  in  Fatigue.     A  pithed 
frog  is  kept  on  ice  for  about  half-an-hour.     Remove  one  hind  limb 
and  replace  it  on  the  ice.     Expose  the  lumbar  plexus  of  the  other 
side  and  stimulate  it  electrically  by  means  of  a  strong  interrupted 
current  for  at  least  ten  minutes.     Cut  off  the  hind  limb,  strip  the 


CH.    IV.]  SARCOLACTIC    ACID.  81 

skin  off  the  two  limbs  and  treat  the  muscles  separately  as  follows  : 
Rapidly  remove  the  muscles,  grind  them  with  ice  cold  95  per  cent, 
alcohol  and  sand.  Transfer  the  mixture  to  a  beaker,  and  warm  for 
a  few  minutes  on  the  water  bath.  Filter  through  a  small  paper 
and  evaporate  to  complete  dryness  on  a  water  bath.  Treat  the 
residue  with  about  5  c.c.  of  cold  water  and  rub  it  up  thoroughly 
with  a  glass  rod.  Filter  and  boil  the  nitrate  with  as  much  animal 
charcoal  as  will  lie  on  a  threepenny  piece.  Filter  and  evaporate 
the  filtrate  to  complete  dryness  on  a  water  bath.  Allow  the  residue 
to  cool  and  apply  Hopkins'  test  by  treating  the  residue  with  strong 
sulphuric  acid,  shaking  round  till  solution  is  obtained,  transferring 
to  a  dry  test  tube,  adding  three  drops  of  saturated  copper  sulphate 
etc.  A  fine  red  colour  develops  in  the  tube  containing  the  extract 
from  the  tetanised  muscle,  but  none  or  very  little  in  the  other. 

Glycogen.  The  percentage  of  glycogen  in  fresh 
muscle  varies  from  0-5  to  1  per  cent.,  so  that  the  total 
amount  in  all  the  muscles  of  the  body  may  be  greater 
than  in  the  liver.  The  muscle  glycogen  decreases  after 
muscular  exercise,  but  not  so  rapidly  as  that  in  the  liver. 

The  estimation  of  glycogen  is  described  on  page  49. 


CHAPTER  V. 

THE   COMPOSITION  OF  THE  DIGESTIVE  JUICES 
AND   THE  ACTION   OF   CERTAIN   ENZYMES. 

The  digestive  enzymes  or  ferments  are  bodies  that 
have  the  power  of  accelerating  the  rate  of  hydrolysis  of 
certain  substances.  They  are  often  divided  into  groups 
depending  on  the  nature  of  the  substance  on  which  they 
act  (the  so-called  substrate  or  zymolyte).  'Thus  those 
acting  on  starch  are  called  amylolytic ;  on  proteins, 
proteolytic  ;  on  fats,  lipolytic,  etc.  The  enzymes  are  often 
named  in  such  a  way  as  to  indicate  their  origin  and  their 
action,  the  termination  -ase  being  employed.  Thus 
ptyalin,  the  amylolytic  enzyme  of  saliva,  can  be  termed 
salivary  amylase,  to  distinguish  it  from  pancreatic  amylase 
(amylopsin).  Gastric  lipase,  the  lipolytic  enzyme  of  the 
gastric  juice,  is  similarly  distinguished  from  pancreatic 
lipase  (steapsin). 

The  chemical  composition  of  the  enzymes  is  at 
present  uncertain,  owing  to  the  extreme  difficulty  of 
preparing  them  in  a  pure  state.  The  proteolytic  enzymes 
are  either  proteins,  or  compounds  so  readily  absorbed  by 
proteins  that  it  is  impossible  to  separate  them.  The 
enzymes  acting  on  certain  of  the  carbohydrates  are 
possibly  themselves  of  a  carbohydrate  nature. 

The  properties  of  the  enzymes  as  a  class  are  as 
follows:  They  are  soluble  in  water,  dilute  salt  solutions, 
dilute  alcohol  and  glycerine.  They  are  precipitated  by 
saturation  with  ammonium  sulphate  and  by  strong 


CH.    V.]  ENZYMES.  83 

alcohol.  They  are  colloidal  and  non-diffusible.  They  are 
most  active  at  a  certain  temperature,  called  the  optimum 
temperature,  which  is  generally  about  45° C.  Their  action 
is  suspended  by  cooling,  but  is  completely  destroyed  by 
raising  the  temperature  to  100°  C. 

The  enzymes  are  remarkably  specific  in  their  action, 
that  is,  they  act  only  on  a  particular  substance  or  on  a 
group  of  substances  having  some  similarity  in  chemical 
composition  and  configuration.  A  striking  example  of 
this  is  seen  in  the  case  of  the  glucosides  (see  page  33). 
The  enzyme  maltase  (a-glucase)  hydrolyses  a-methyl-  and 
a-ethyl-d-glucosides,  but  has  no  action  on  /3-methyl-  or 
/3-ethyM-glucosides,  or  on  any  Z-glucoside  or  on  d-  or 
Z-galactosides.  The  enzyme  emulsin  (/3-glucase)  acts  only 
on  /3-ethyl,  methyl  or  phenyl-<i-glucosides.  Lactase  acts 
only  on  the  /3-galactosides.  It  is  probable  that  the  enzyme 
first  unites  with  the  substrate,  and  to  do  this  it  must  have 
a  configuration  in  space  corresponding  with  that  of  the 
substrate. 

The  hydrolysis  is  effected  by  the  water  molecules,  or 
by  the  H  and  OH  ions  formed  from  the  water.  In  some 
cajses  a  certain  concentration  of  H  or  OH  ions  must  be 
present  to  enable  the  enzyme  to  act.  Thus  pepsin  acts 
in  acid  solution  only :  trypsin  requires  a  certain  concentra- 
tion of  OH  ions. 

The  action  of  most  enzymes  is  retarded  by  the 
accumulation  of  the  products  of  the  reaction,  and  in 
certain  cases  the  reaction  is  reversible. 

This  is  well  seen  in  the  case  of  lipase,  which  induces 
the  following  reaction  : — 

Ethyl  butyrate  -f  water  <  >  ethyl  alcohol  +  butyric  acid. 
The  velocity  of  reaction  is  proportional  to  the  amount  of 
the  enzyme  present,  provided  that  the  amount  of  the 


84  COMPOSITION     OF    THE     DIGESTIVE    JUICES.         [CH.    V. 

enzyme  is  very  small  compared  with  that  of  the  substrate. 
If  the  amounts  of  enzyme  and  substrate  are  at  all  com- 
parable, the  laws  of  mass  action  are  followed.  But  com- 
plications are  introduced  by  the  fact  that  some  of  the 
enzyme  is  thrown  out  of  action  by  being  absorbed  by  the 
products  of  the  action. 

In  certain  cases  enzyme  action  is  dependent  on  the 
simultaneous  presence  of  two  substances.  These  are 
sometimes  called  co-ferments.  It  has  been  shewn  that 
the  zymase  that  is  responsible  for  the  alcoholic  fermenta- 
tion of  sugar  by  yeast  can  only  act  in  co-operation  with 
phosphates  and  some  substance  that  is  diffusible  and  not 
destroyed  by  boiling.  Also  the  lipase  of  the  pancreas 
requires  the  presence  of  some  soluble,  heat-stable  sub- 
stance to  allow  it  to  act.  Bile  salts  have  this  property,  as 
has  been  seen  in  a  previous  chapter.  The  action  of  the 
enzymes  can  be  retarded  by  certain  substances.  These 
are  of  two  classes :  paralysers  and  anti-enzymes.  The 
paralysers  are  generally  salts  of  the  heavy  metals,  which 
probably  alter  the  physical  state  of  the  colloidal  enzymes. 
The  anti-enzymes  are  of  an  organic  nature.  They  probably 
combine  with  the  enzyme  and  thus  prevent  it  from 
acting  on  the  substrate.  Examples  are  seen  in  the  case 
of  the  anti-trypsin  of  normal  serum,  of  the  intestinal 
mucous  membrane  and  of  the  tissues  of  intestinal 
parasitic  worms. 

A.     Saliva. 

Saliva  is  of  value  as  a  lubricant  in  the  act  of  degluti- 
tion, and  in  some  animals  this  is  its  sole  function.  In 
many  animals,  however,  it  contains  an  enzyme,  ptyalin, 
which  acts  on  starch,  converting  it  finally  into  maltose, 
with  perhaps  a  small  amount  of  glucose.  It  is  claimed  by 
certain  workers  that  for  the  complete  hydrolysis  of  starch 
three  ferments  are  necessary,  viz.,  amylase  that  converts 


CH.    V.]  PTYALIN.  85 

starch  into  the  dextrins  :  dextrin ase  that  converts  the 
dextrins  into  maltose :  and  maltase  that  converts  maltose 
into  glucose.  In  the  case  of  the  action  of  ptyalin  on  starch 
as  conducted  in  vitro,  the  final  product  consists  of  about 
80%  of  maltose,  the  remaining  20%  being  a  comparatively 
simple  dextrin  called  "  stable  dextrin,"  owing  to  its 
resistance  to  the  further  action  of  the  ferment.  But  if 
this  dextrin  be  isolated  the  action  of  ptyalin  is  to 
hydrolyse  it  very  slowly  and  incompletely  to  equal  parts 
of  maltose  and  glucose. 

Ptyalin  acts  best  in  a  medium  that  is  very  faintly 
acid.  It  is  rapidly  destroyed  by  dilute  HC1,  but  can  be 
protected  by  the  presence  of  proteins  with  which  the  acid 
combines,  the  concentration  of  hydrogen  ions  being  thus 
decreased.  It  is  probable  that  the  action  of  ptyalin  on  the 
carbohydrates  of  a  mixed  meal  continues  for  about  30 
minutes  in  the  mixed  gastric  contents. 

Inorganic  salts,  particularly  sodium  chloride,  favour 
its  action,  probably  by  causing  the  appearance  of  hydrogen 
ions,  by  some  obscure  absorption  phenomenon  of  the 
colloidal  starch.  This  effect  of  NaCl  is  best  seen  if  the 
ferment  preparation  has  previously  been  freed  from 
electrolytes  by  alcohol  precipitation  and  thorough  dialysis 
against  distilled  water.  Such  preparations  are  almost 
inactive,  but  become  active  on  the  addition  of  traces  of 
weak  acids  or  neutral  salts. 

174.  Collect  about  5  c.c.  of  your  own  saliva  in  a  small  beaker. 
Test  the  reaction  with  neutral  litmus  paper :   it  is  alkaline. 

NOTE.— The  first  portion  of  saliva  collected  is  very  apt  to  be  neutral 
or  even  slightly  acid,  probably  owing  to  bacterial  decomposition  in  the  mouth. 
But  if  the  secretion  is  free,  that  collected  later  is  invariably  alkaline. 

175.  Transfer  the  saliva  to  a  test  tube  and  add  strong  acetic 
acid.     A  stringy  precipitate  of  mucin  is  formed,  insoluble  in  excess 
of  acid.     Stir  the  mixture  vigorously  with  a  glass  rod  :  the  mucin 


86  COMPOSITION     OF    THE     DIGESTIVE    JUICES.         [CH.   V. 

forms  a  clump  which  can  be  removed  by  the  rod.  To  the  clear 
fluid  remaining  add  some  Millon's  reagent  and  boil.  Only  a  slight 
red  precipitate  is  formed,  showing  that  the  proteins  of  saliva 
consist  almost  entirely  of  mucin. 

176.  Obtain   diluted   saliva  as  follows  :  warm  some  distilled 
water  in  a  beaker  to  about  40°  C.    With  a  portion  of  this  thoroughly 
rinse  the  mouth  out.     iSfow  take  about  20  c.c.  of  the  warm  water 
into    the    mouth    and    move   it    about  by   the   tongue   for  at  least 
a  minute.     Collect  the  fluid  thus  obtained  in  a  clean  beaker,  and 
repeat  the  process  twice  more.     Thoroughly  mix  the  diluted  saliva 
thus  obtained,  shake  it  vigorously  and  filter. 

177.  In  a  clean  test  tube  place  5  c.c.  of    1   per  cent,  starch 
paste,  freshly  prepared  with  distilled  water,  and  5  c.c.  bf  the  diluted 
saliva.     Mix  well  and  place  the  tube  in  a  water-bath  maintained  at 
a   temperature  of  about  40°  C.     Place  a  series  of  drops  of  iodine 
solution  on  a  white  porcelain  plate,  and  from  time  to  time  transfer, 
by  means  of   a  glass  rod,  a   drop  of  the  digesting  mixture   to   a 
drop  of  the  iodine.     The  blue  colour  produced  at  first  will  later 
become  blue-violet,  red-violet,  red-brown,  and  light-brown  yellow, 
as    the    starch,    and   then    the    erythro-dextrin  are   converted   into 
other  products.     When  a  drop  of  the  mixture  no  longer  gives  any 
colour  with  iodine,  boil  a  few  c.c.  of  it  with  a  few  c.c.  of  Fehling's 
solution.     A    well-marked    reduction    is    obtained,   showing  that  a 
ferment  (ptyalin)  in  saliva  has  converted  the  starch  into  a  reducing 
sugar,  which  is,  however,  not  glucose,  but  maltose. 

178.  Perform  a  control  test  by  first  boiling,  and  then  cooling 
the  saliva  before  adding  it  to  the  starch.    (See  Ex.  118.)     No  action 
whatever  takes  place  when  the  mixture  is  allowed  to  stand  on  the 
warm  bath,  proving  that  the  effect  in  the  above  exercise  was  due  to 
a  ferment. 

179.  The  investigation  of  the  activity  of  ptyalin  under 
various  conditions  by  the  method  of  the  achromic  point. 

In  each  of  a  series  of  clean  test  tubes  place  about  1  c.c.  of  an 


CH.   V.]  PTYALIN.  87 

iodine  solution  that  has  been  diluted  to  a  pale  straw-colour  with 
distilled  water. 

Carefully  measure  5  c.c.  of  the  1  per  cent,  starch  paste  into  a 
perfectly  clean  test  tube,  add  five  drops  of  distilled  water  and  place 
the  tube  in  a  warm  bath  at  40°  C.  for  a  few  minutes. 

To  the  starch  paste  add  5  c.c.  of  the  difoted  saliva,  previously 
warmed  to  40°  C.  in  the  warm  bath.  Mix  the  two  fluids,  and  note 
the  time  of  the  addition.  At  intervals  transfer  a  few  drops  of  the 
digestive  mixture  to  one  of  the  samples  of  iodine  by  means  of 
a  small  pipette  made  from  quill  tubing.  The  same  series  of  colour 
changes  will  be  observed  as  were  seen  in  Ex.  177.  Note  the  time 
when  the  addition  of  the  mixture  of  iodine  ceases  to  produce  any 
colour.  This  point,  which  is  the  moment  when  the  last  trace  of 
erythro-dextrin  is  converted  into  achroo-dextrin  and  maltose,  is 
known  as  the  achromic  point.  The  time  that  is  taken  to  reach  this 
point  ("chromic  period")  is  a  measure  of  the  activity  of  the  ferment. 

Repeat  the  exercise,  and  note  that  the  chromic  period  obtained 
agrees  fairly  closely  with  that  previously  found. 

NOTES. — 1.  It  will  be  seen  that  the  starch  and  the  ferment  solution  are 
separately  warmed  to  the  temperature  at  which  the  exercise  is  performed. 
Otherwise  the  results  will  not  be  strictly  comparable  with  those  of  the  following 
exercises. 

2.  A  convenient  chromic  period  is  one  of  about  five  minutes.  If  it  is  less 
than  two  minutes,  the  saliva  should  be  diluted  with  an  equal  volume  of  distilled 
water.  If  it  is  more  than  ten  minutes  the  starch  paste  should  be  diluted  with  an 
equal  bulk  of  water  and  boiled  well  to  ensure  thorough  mixing.  In  either  case 
the  chromic  period  under  these  new  conditions  must  be  carefully  noted  for 
comparison  with  those  obtained  in  the  following  exercises. 

180.  Repeat  the  above  exercise,  substituting  five  drops  of 
a  5  per  cent,  solution  of  sodium  chloride  for  five  drops  of  water. 
The  chromic  period  is  considerably  reduced. 

NOTE. — The  concentration  of  NaCl  in  the  digestive  mixture  is  between  '01 
and  -02  per  cent.  Even  lower  concentrations  than  this  have  a  marked  effect  in 
increasing  the  activity  of  ptyalin.  A  concentration  of  5  per  cent,  of  NaCl 
usually  slightly  decreases  the  activity. 


88  COMPOSITION     OF    THE     DIGESTIVE    JUICES.         [CH.    V. 

181.  Repeat  the  above  exercise,  using  one  drop  of  '4  per  cent, 
hydrochloric  acid  and  four  drops  of  water.     The  chromic'  period  is 
considerably  reduced.     With  two  drops  of  '4  per  cent.  HC1  and 
three  drops  of  water  the  chromic  period  may  or  may  not  be  reduced, 
according  to  the  alkalinity  of  the  saliva. 

NOTE.— One  drop  of  -4  per  cent.  HC1  in  the  mixture  gives  a  concentration 
of  acid  of  about  '002  per  cent.  But  the  saliva  contains  a  little  alkali  and  also 
some  protein  which  enters  into  a  loose  combination  with  the  acid.  The  con- 
centration of  free  HC1  in  the  digestion  mixture  will  therefore  be  less  than 
•002  per  cent. 

182.  Repeat    the  above  exercise,  using  five  drops  of  '4  per 
cent,  hydrochloric  acid  instead  of  one  drop.     The  chromic  period  is 
indefinitely  prolonged. 

NOTE. — The  concentration  of  HC1  in  this  experiment  is  undjer  '02  per  cent. 
In  the  absence  of  proteins  such  a  concentration  rapidly  destroys  ptyalin,  the 
activity  not  returning  on  neutralisation. 

183.  Repeat  Exercise  179  at  the  temperature  of  the  room,  at 
30°  C.  and  at  55°  C.     The  chromic  period  is  least  at  45°  C. 

B.     Pepsin. 

Pepsin  is  the  proteolytic  ferment  found  in  the  gastric 
juice.  It  acts  on  most  proteins,  finally  converting  them 
into  a  mixture  of  peptones  and  polypeptides.  It  is  impor- 
tant to  note  that  it  does  not  hydrolyse  them  as  far  as  free 
amino-acids,  thus  differing  from  trypsin  and  erepsin. 
The  intermediate  stages  in  the  action  are  given  on 
page  24. 

Pepsin  acts  in  an  acid  medium  only.  The  optimum 
strength  of  acid  is  one  with  a  concentration  of  hydrogen 
ions  found  in  a  O2  per  cent,  solution  of  HC1.  The  fer- 
ment is  rapidly  destroyed  by  alkalies.  It  is  secreted  by 
the  peptic  cells  of  all  parts  of  the  stomach,  in  which 
it  appears  as  a  precursor,  called  pepsinogen.  This  is 
relatively  stable  to  alkalies  and  is  converted  into  pepsin 
by  the  action  of  HC1. 


CH.    V.]  PEPSIN.  89 

For  the   following   experiments   use    a    1    per    cent, 
solution  of  commercial  pepsin  in  water. 

184.  Place  equal  amounts  of  fresh  washed  fibrin  in  four  test 
tubes  labelled  A,  B,  C,  and  D. 

To  A  add  5  c.c.  of  pepsin  and  5  c.c.  of  -4  per  cent.  HC1. 
To  B  add  5  c.c.  of  pepsin  and  5  c.c.  of  water. 
To  C  add  5  c.c.  of  water  and  5  c.c.  of  '4  per  cent.  HC1. 
To  D  add  5  c.c.  of  pepsin  that  has  been  boiled  and  then  cooled, 
and  5  c.c.  of  -4  per  cent.  HC1. 

Place  the  four    tubes  in  a  water  bath  at  40°  C.  for   at  least 
thirty  minutes. 

Note  that  in 

A,  the  fibrin  swells  up,  becomes  transparent  and  then  dissolves  ; 

B,  the  fibrin  is  unaltered ; 

C,  the   fibrin    swells    up,    becomes  transparent,  but  does  not 
dissolve ; 

D,  the  fibrin  is  like  that  in  C. 

NOTE. — These  exercises  show  that  neither  -2  per  cent.  HC1  alone,  nor 
pepsin  alone,  can  digest  fibrin,  but  that  pepsin  in  the  presence  of  '2  per  cent. 
HC1  has  this  property.  In  D  the  ferment  pepsin  has  been  destroyed  by  boiling. 

185.  The  detection  of  pepsin.     Obtain  some  fibrin  that  has 
been    stained  with  carmine  (see  note  below).     Treat  the  ferment 
solution  with  the  same  volume  of  0*4  per  cent.   HC1.     Divide  this 
into  two  equal  portions  and  label  them  A  and  B.     Boil  B  for  a 
minute,  and  cool  the  tube.     To  each  tube  add  a  few  flakes  of  the 
stained    fibrin.     Place    them    on    the  warm  bath  for  ten  minutes. 
Shake  and  observe  the  colour  of  the  fluid.     In  A  it  will  be  red.     In 
B  it  will  be  almost  or  quite  colourless. 

NOTE.— The  carmine  solution  for  staining  fibrin  is  prepared  by  dissolving 
1  gram  of  carmine  in  about  1  c.c.  of  ammonia  and  adding  400  c.c.  of  water. 
The  solution  is  kept  in  a  loosely-stoppered  bottle  till  the  smell  of  ammonia  has 
become  faint.  Fresh  washed  fibrin  is  chopped  finely,  placed  in  the  carmine 
solution  for  twenty-four  hours,  strained  off  and  washed  in  running  water  till  the 
washings  are  colourless.  If  not  required  immediately,  it  should  be  kept  under 
ether  and  washed  with  water  before  use.  It  cannot  be  used  for  testing  for 
trypsin,  owing  to  the  solubility  of  the  dye  in  alkalies. 


90  COMPOSITION     OF    THE     DIGESTIVE    JUICES.         [cH.    V. 

186.  The  estimation  of  Pepsin  by  Mett's  method. 

Preparation  of  the  tubes.  The  whites  of  several  new-laid 
eggs  are  beaten  to  break  the  membranes,  strained  through  linen 
or  muslin  and  allowed  to  stand  till  free  from  air  bubbles.  The 
liquid  is  then  drawn  up  into  lengths  of  glass  tubing  with  an  internal 
diameter  of  between  1  and  3  mm.  Each  length  is  laid  flat  on  a  piece 
of  wire  gauze,  so  arranged  that  it  can  be  dropped  into  a  saucepan  of 
hot  water,  having  a  double  bottom  ("porridge  saucepan").  The  water 
in  the  saucepan  is  boiled  and  allowed  to  stand  till  that  in  the  inner 
vessel  has  cooled  to  85°  C.  The  gauze  with  the  prepared  tubes  is 
then  placed  in  this  inner  vessel,  and  allowed  to  stand  till  the  water 
is  quite  cold.  The  tubes  can  be  preserved  by  sealing  the  ends  with 
shellac. 

Method  of  estimation.  Cut  off  lengths  of  2  cms.,  breaking  the 
tubes  sharply  to  get  an  even  edge  of  coagulated  egg  white. 

Measure  10  to  20  c.c.  of  the  ferment  into  a  small  Erlenmeyer 
flask.  In  it  place  three  of  the  tubes  of  egg-white,  shake  and  cork, 
and  place  the  flask  in  a  thermostat  at  40°  C.  for  24  hours.  The 
mixture  must  not  be  shaken  during  the  digestion.  Measure  the 
length  of  the  tube  (T)  and  of  the  remaining  egg-white  (W)  by 
means  of  a  millimetre  scale  and  a  magnifying  glass.  T  — W  =  the 
amount  of  protein  digested  (D).  Take  the  average  for  the  three 
tubes.  D  varies  as  the  square  root  of  the  amount  of  ferment  present. 

NOTES. — Filtered  gastric  contents  should  be  diluted  with  —  HC1    in   the 

30 
proportion  of  1  c.c.  of  gastric  contents  to  15  c.c.  of  acid. 

N 
For  practice  use  a  0'5  per  cent,  solution  of  Merck's  pepsin  injQ  HC1. 

Dilute  1,  4,  and  9  c.c.  to  16  c.c.  with  —  HC1.  The  amounts  of  egg-white 
digested  should  be  as  v'l  :  v  4  :  V9  i.e.  as  1  :  2  :  3. 

187.  Action  of  alkalies  on  Pepsin.     Treat   5   c.c.   of  the 
pepsin  solution  with  half  its  volume  of  2  per  cent,  sodium  carbonate 
and  place  on  the  bath  at  40°  C.  for  half-an-hour.     Neutralise  with 
•4  per  cent.  HC1,  and  then  add  an  equal  volume  of  -4  per  cent. 


CH.    V.J  ACIDITY    OF    GASTRIC    JUICE.  91 

HC1  to  the  fluid.  Add  some  carmine  fibrin  and  place  the  tube  on 
the  warm  bath.  The  fibrin  does  not  dissolve,  showing  that  pepsin 
is  destroyed  by  dilute  alkaline  salts. 

C.     The  Acidity  of  Gastric  Juice. 

The  acidity  of  the  gastric  contents  is  due  to  three 
causes,  viz.: 

1.  The  free  hydrochloric  acid. 

2.  The  HC1  combined  with  proteins. 

3.  Acid  salts. 

The  sum  of  these  three  is  called 

» 

4.  The  total  acidity. 

The  sum  of  1  and  2  is  called 

5.  The  physiologically  active  HC1. 

The  estimation  of  these  different  quantities  in  the 
gastric  contents  is  of  considerable  importance  in  many 
pathological  conditions.  A  test  meal  of  toast  and  tea  is 
given,  and  an  hour  afterwards  the  gastric  contents  are 
removed  by  means  of  a  tube. 

Total  acidity.  Ten  c.c.  of  the  filtered  contents  are 
titrated  with  N/10  NaOH,  using  phenolphthalein  as  an 
indicator.  The  result  is  expressed  in  terms  of  grams  of 
HC1  in  100  c.c.,  by  multiplying  the  number  of  c.c.  by 
0-0365. 

Free  HC1.  The  estimation  of  this  is  practically  that 
of  the  concentration  of  hydrogen  ions  in  the  gastric 
contents.  HC1  is  very  freely  dissociated  into  H  and  Cl 
ions  in  such  dilutions  as  those  found  in  the  stomach. 
But  weak  acids,  as  lactic  and  butyric,  are  only  slightly 
dissociated.  Also  the  addition  of  proteins  to  a  solution  of 
HC1  decreases  the  concentration  of  H  ions,  owing  to  the 
formation  of  a  compound  that  only  dissociates  to  a 


92  COMPOSITION     OF    THE     DIGESTIVE    JUICES.        [CH.    V. 

relatively  small  extent.  The  student  is  advised  to  read 
the  remarks  on  acidity  in  the  section  on  the  acidity  of  the 
urine. 

The  estimation  of  the  free  HC1  is  hest  done  by  the 
electrical  method  that  is  mentioned  in  the  section  quoted 
above. 

The  use  of  indicators  is  not  to  be  advised.  According 
to  the  latest  researches  it  is  certain  that  even  Toepfer's 
reagent  (dimethyl-amido-azo-benzene)  reacts  with  an 
excess  of  butyric  and  lactic  acids,  and  also  with  HC1  in 
combination  with  protein. 

The  simplest  clinical  method  that  gives  results  at  all 
comparable  with  the  electrical  method  is  that  of  titrating 
with  standard  NaOH  until  no  reaction  is  obtained  for 
free  HC1  with  Gunsberg's  reagent.  The  method  is  rather 
tedious. 

187 A.     Gunsberg's  test  for  free  hydrochloric  acid. 

A.  Place  a  single  drop  of  Gunsberg's  reagent  in  a   porcelain 
dish  on  a  boiling  water  bath.     When   dry,  add  a  single  drop  of 
0'04  per  cent.  HC1  to  the  film  of  reagent  and  again  take  to  dryness. 
A  brilliant  carmine  colour  develops. 

B.  Repeat  the  experiment,  using  a  mixture  of  equal  parts  of 
1  per  cent,  acetic  acid  and  1  per  cent,  sodium  chloride  in  place  of 
the  HC1.     Only  a  yellow  or  brown  stain  results. 

C.  To  10  c.c.  of  0-04  per  cent.  HC1  add  5  c.c.  of   1   per  cent. 
Witte's  peptone.     Try  Gunsberg's  test  with  a  drop  of  this.     Free 
HC1  is  absent. 

D.  To  the  remainder  of  the  fluid  C  add  a  drop  of  phenol  - 
phthalein    and    titrate  with  N/10   NaOH  till  pink.     Compare  the 
amount   used  with  that  required  to  neutralise  10  c.c.  of  0*04  per 
cent.    HC1.      The  absence  of  free  HC1  in  C  is  obviously  not  due 
to   the    presence    of    any    alkali    in    the    peptone.     The    HC1    has 
combined  with  the  protein  to  form  a  protein- HC1  compound. 


CH.    V.J  ACIDITY    OF    GASTRIC    JUICE.  93 

NOTE.— Preparation  of  the  reagent.  Dissolve  2  grammes  of  phloro- 
glucin  and  1  gramme  of  vanillin  in  30  c.c.  of  absolute  alcohol.  The  solution 
should  be  freshly  prepared  to  give  sharp  results,  but  it  can  be  preserved  for  a 
certain  time  in  dark  glass  bottles. 

188.  Estimation  of  free  HC1  by  Gunsberg's  reagent.     If 

free  HC1  is  present  as  determined  by  the  method  given  in  the 
previous  exercise,  titrate  10  c.c.  of  the  fluid  with  N/10  soda, 
performing  Gunsberg's  test  with  a  drop  of  the  mixture  after  every 
addition.  The  end  point  is  reached  when  a  drop  of  the  mixture 
fails  to  give  the  test.  If  many  drops  have  been  used,  the  titration 
must  be  repeated,  adding  nearly  the  whole  of  the  calculated  amount 
of  soda  in  one  operation. 

Calculation.     Multiply  the   amount    of    N/10    soda   used    by 
0'0365.     The  result  is  the  number  of  grams  of  free  HC1  per  100  c.c. 

189.  Prout- Winter   method   for  the    estimation  of  the 
physiologically  active  HC1  and  of  mineral  chlorides. 

A.  10  c.c.  of  the  filtered  gastric  contents  are  mixed  with  an 
excess  of  sodium  bicarbonate  in  a  platinum  crucible  and  evaporated 
to  dryness  over  a  water-bath.     The  crucible  is  then  heated  over 
a  Bunsen  flame  and  the  contents  incinerated.     The  total  chlorides 
in  the  ash  is  determined  by  extracting  with  water  and  applying 
Volhard's  method.     Express  the  result  in  terms  of  HC1  per  100  c.c. 

B.  Repeat  the   experiment  without  adding  the  bicarbonate. 
The  free  HC1  and  that  combined  with  proteins  is  evolved,  and  only 
the  mineral  chlorides  retained.     Estimate  these  as  before.     A  minus 
B  gives  the  amount  of  physiologically  active  HC1. 

NOTE. — Usually  the  "active"  HC1  is  only  slightly  less  than  the  total 
acidity,  shewing  that  no  abnormal  acids  are  present.  But  in  certain  diseases 
there  is  a  great  difference  between  the  two  results,  and  it  is  in  these  cases 
that  the  estimation  is  of  value. 

The  amount  of  mineral  (sodium)  chloride  is  of  great  interest  in  connection 
with  carcinoma,  in  which  condition  free  HC1  is  absent  and  the  mineral  chlorides 
are  much  increased.  This  may  point  to  a  neutralisation  of  the  acid  by  some 
alkaline  secretion. 

In  gastric  ulcer  the  free  HC1  is  increased  above  normal,  and  is  always 
considerably  greater  than  the  mineral  chlorides. 


94  COMPOSITION     OF    THE     DIGESTIVE    JUICES.        [CH.    V. 

D.     Trypsin. 

Trypsin  is  the  proteolytic  ferment  secreted  by  the 
pancreas.  The  pancreatic  juice  contains  a  precursor 
called  trypsinogen.  This  is  converted  into  trypsin  011 
reaching  the  duodenum  by  the  action  of  the  enterokinase 
secreted  by  the  mucous  membrane  of  the  small  intestine. 

Trypsin  differs  from  pepsin  in  two  important  par- 
ticulars. In  the  first  place  it  acts  in  a  medium  that 
is  alkaline  to  litmus.  The  optimum  concentration  of 
hydroxyl  ions  is  not  certain.  Probably  that  concentration 
in  which  the  ferment  acts  best  is  one  that  has  a  destruc- 
tive action  on  the  ferment.  Consequently  the  optimum 
concentration  of  alkali  will  be  greater  for  a  "short  than 
for  a  long  digestion.  It  is  important  to  note  in  this 
connection  that  trypsin  is  not  at  all  stable  in  alkaline 
solutions.  To  preserve  the  ferment  a  minute  amount  of 
acid  is  added. 

In  the  second  place  trypsin  differs  from  pepsin  in  being 
able  to  hydrolyse  the  protein  molecule  to  the  final 
products,  the  various  amino-acids  and  basic  substances. 

Preparation  of  trypsin.  Obtain  the  fresh  pancreas  of  a  pig.  Free  it 
from  fat  as  far  as  possible.  Weigh  it.  Mince  it  finely  and  add  three 
times  its  weight  of  distilled  water  and  its  own  weight  of  strong  alcohol. 
Shake  well  in  a  flask  and  allow  it  to  stand  for  three  days  at  room  tempera- 
ture, shaking  the  flask  occasionally.  Strain  through  muslin  and  filter 
through  a  large  folded  filter.  The  filtrate,  which  comes  through  very 
slowly,  is  measured  and  treated  with  1  c.c.  of  strong  HC1  for  every  litre. 
This  causes  the  appearance  of  a  cloudy  precipitate,  which  settles  in  a 
week  or  so  and  can  then  be  filtered  off.  The  fluid  keeps  for  an  indefinite 
period,  if  stoppered,  without  the  addition  of  any  antiseptic,  the  alcohol 
itself  acting  as  an  antiseptic.  The  fluid  is  rich  in  trypsin  and  in 
amylopsin,  the  amylolytic  ferment  of  the  pancreas.  It  does  not  contain 
any  lipase. 

It  seems  to  be  identical  with,  though  usually  rather  more  active  than, 
the  commercial  extract  known  as  Benger's  "liquor  pancreaticus." 


CH.    V.J  DETECTION     OF    TRYPSIN.  95 

Detection  of  Trypsin. 

The  digestion  of  fibrin  does  not  give  a  satisfactory 
method  for  the  determination  of  the  presence  of  trypsin 
owing  to  the  relatively  slow  rate  at  which  the  action 
takes  place. 

The  best  method  is  that  of  Gross,  who  uses  a  solution 
of  casein.  This  is  precipitated  by  dilute  acetic  acid,  but 
it  is  rapidly  acted  on  by  trypsin  and  is  converted  into 
substances  that  are  soluble  in  dilute  acids.  We  thus  have 
a  means  both  of  detecting  and  of  comparing  the  activities 
of  tryptic  solutions,  by  finding  the  time  required  for  the 
disappearance  of  a  certain  amount  of  casein. 

Preparation  of  the  Casein  Solution.  Dissolve  5  grams  of  Haminarsten's 
casein  in  42'5  c.c.  of  N/10  NaOH  and  450  c.c.  of  boiling  water.  Filter 
Vhilst  still  warm,  cool  and  make  the  volume  up  to  500  c.c. 

190.  Measure  10  c.c.  of  the  casein  solution  into  a  test  tube 
and  place  it  on  the  warm  bath  for  a  few  minutes,  so  that  it  may 
acquire  the  temperature  of  the  bath. 

Measure  5  c.c.  of  the  pancreatic  extract  (previously  diluted 
with  four  volumes  of  water)  into  another  tube  and  warm.  Mix 
the  two  solutions,  noting  the  time.  At  intervals  remove  about  a  c.c. 
by  means  of  a  pipette  or  glass  tube  and  run  it  into  a  similar  volume 
of  1  per  cent,  acetic  acid.  At  first  a  heavy  white  precipitate  of 
casein  is  produced.  But  after  a  certain  length  of  digestion,  de- 
pending on  the  activity  of  the  ferment,  no  precipitate  is  produced. 

NOTE. — The  disappearance  of  the  casein  cannot  be  due  to  pepsin,  for  free 
HC1  is  not  present. 

The  products  of  the  action  of  Trypsin  on  Proteins. 

The  final  products  of  the  action  of  trypsin  and  other 
powerful  hydrolytic  reagents  on  proteins  consist  of  a 
number  of  substances  which  differ  somewhat  in  nature 
and  amount  with  the  protein.  They  are  mostly  mon- 
.amino  acids,  with  the  amino-group  replacing  an  H  atom 


96    *  COMPOSITION    OF    THE    DIGESTIVE    JUICES.         [CH.    V. 

attached  to  that  carbon  atom  which  is  itself  attached  to 
the  -COOH  group.     That  is,  they  are  a-amino  acids. 

CH3.CH2.COOH.     Propionic  acid. 
CH3CH(NH2).COOH.    a-amino-propionic  acid. 
CH2(NH2)CH2.COOH.    /3-amino-propioiiic  acid. 

Classification  of  the  Products. 

..,..  ,.      (  Fatty  series.  Group  A. 

Mon-     f.Mono-carboxyhc.  )  J 

amino-  ^  '  Aromatic  series.    Group  B. 

acids.    (  Di-carboxylic.      Group  C. 
Di-amino-acids.  Group  D. 

Heterocyclic  compounds.     Group  E. 

Carbohydrate  compound.    Glucosamiiie,  an  amino-hexose. 
Group  A.       1.     Glycine  (amino-acetic  acid),  CH2.(NH2).COOH. 

2.  Alanine   (a-amino-propionic  acid),  CH3.CH.(NH2).COOH. 

3.  Leucine  (a-amino-isocaproic  acid),  C6Hi3NO2. 

4.  Cystine  (dicysteine,  or  di-/?-thio-a-amino-propionic  acid). 
Group  B.      5.     Phenylalanine.       C6H5.CH2.CH.(NH2).COOH. 

6.     Tyrosine  (oxy-phenyl-alanine), 

OH 

.  COOH  . 


7.     Tryptophane  (indol-alanine)  , 

C8H6N.CH2.CH.(NH2).COOH. 

Group  C.      8.    Aspartic  acid  (amino-succinic  acid). 

9.     Glutamic  acid  (a-amino-glutaric  acid). 
Group  D.     10.     Arginine  (a-amino-8-guanidine-valerianic  acid), 

TTXT    n^-^'NHa 
N  :  °-^^NH.   CH2.CH2.CH2.CH.(NH2).COOH. 

11.     Lysine  (a,  e-diamino-caproic  acid). 

Group  E.   12.      Histidine  (/?-imidazole-alanine)  , 

CH 

/        \N 

HN         N 
I  I 

CH  -  C  —  CH2.CH.(HN2).COOH. 


CH.    V.]  TRYPTIC     DIGESTION.  97 

13.     Proline  (a-pyrrolidine-carboxylic  acid), 


I  I 

CH2      CH.COOH 

\        / 
NH 

Tryptophane  (indol-alanine) . 

C.CH2.CH(NH2).COOH 

'  CH 


NH. 
The  Isolation  of  the  Products. 

The  following  three  methods  have  been  employed. 

1.  Fractional  crystallisation. 

2.  Fractional  precipitation,  that  is,  a  reagent  is  used  which  only 

precipitates  one  or  two  of  the  substances  present  in  the 
mixture,  e.g.  mercuric  sulphate  in  acid  solution  only  precipi- 
tates tryptophane  and  cystine  :  phosphotungstic  acid  only 
precipitates  Groups  D  and  E  (with  the  exception  of  proline). 

3.  Fractional  distillation  of  the  esters.     The  compounds  are  con- 

verted into  their  ethyl  esters,  which  are  dried  and  distilled 
under  very  low  pressures.  Since  they  have  different  boiling 
points  they  can  be  separated. 

191.  150  grams,  of  commercial  casein  ("  protene "  or 
"  plasmon"),  50  to  100  c.c.  of  the  tryptic  solution  described  on  page  94, 
and  a  litre  of  1  per  cent.  Na2CO3,  have  been  digested  for  about  ten 
days  at  40° C.  in  a  large  flask,  1  gram,  of  sodium  fluoride  and  about 
30  c.c.  of  chloroform  or  tuluol  being  added,  and  the  mouth  of  the 
flask  securely  plugged  with  cotton  wool,  soaked  in  chloroform,  to 
prevent  bacterial  decomposition.  About  100  c.c.  of  the  mixture 
are  given  to  you.  Boil  the  mixture,  and  whilst  boiling  add  strong 
acetic  acid,  drop  by  drop,  till  the  reaction  is  acid.  Cool  under  the 
tap,  and  filter  off  the  undigested  casein,  etc. 

A.  Treat  5  c.c.  of  the  filtrate  with  bromine  water,  drop  by 
drop ;  a  pink  colour  gradually  develops,  which  deepens  and  then 
disappears  as  more  bromine  water  is  added.  When  the  colour  is 
no  longer  intensified  by  the  addition  of  bromine,  add  2  or  3  c.c.  of 
amyl  alcohol  and  shake.  On  standing,  the  alcohol  rises  to  the 

ii 


98  COMPOSITION     OF    THE     DIGESTIVE     JUICES.        [cH.    V. 

surface  coloured  a  fine  red  or  violet.     This  reaction  is  due  to  the 
presence  of  tryptophane. 

B.  Treat    another    5    c.c.    of    the    filtrate    with    ten    drops    of 
concentrated    sulphuric    acid    and    10    c.c.    of    a     10    per    cent, 
solution    of    mercuric    sulphate    in    5    per    cent.     H2SC>4.      Shake 
the    tube    and    leave     it    for    five    minutes.       Note    the     yellow 
precipitate  of  a  mercury  compound  of  tryptophane.     Filter  this  off 
and  label  the  filtrate  A.     Wash  the  precipitate  through  a  hole  in 
the  paper  into  a  clean  tube,  fill  with  water,  shake  and  filter  again? 
neglecting  the  filtrate.       Wash  the  precipitate  on  the  paper  once 
more  with  water  and  then  let  it  drain.     Scrape  a  portion  off  the 
paper,  transfer  it  to  a  tube,  add  2  c.c.  of  "  reduced  oxalic  acid  "  and 
then    2    c.c.   of   concentrated   sulphuric   acid.     A   purple   colour   is 
produced,  showing  that  tryptophane  is  responsible  for  trie  glyoxylic 
reaction.     (See  Ex.  3.) 

Treat  another  portion  of  the  precipitate  with  Millon's  reagent 
and  boil.  A  yellow  colour  is  produced,  not  the  characteristic  red  of 
Millon's  reaction. 

To  another  portion  of  the  precipitate  apply  the  xanthoproteic 
test.  A  well-marked  reaction  is  obtained.  (See  notes  to  Ex.  1.) 

To  portions  of  filtrate  A  apply  the  glyoxylic,  Millon's,  and  the 

xanthoproteic    reactions.     Only   the    latter    two    are   obtained,  the 

tryptophane,    but    not   the  tyrosine,  having   been   removed   by  the 
mercury  reagent  employed. 

C.  Treat  the  remaining  90  c.c.  of  the  filtrate  with  a  few  drops 
of  ammonia  and  evaporate  to  a  small  bulk  (about  20  c.c.)  either  on 
the  water-bath  or  by  use  of  a  small  free  flame.     Allow  the  residue 
to  stand  twenty -four  hours.     Notice  the  formation  of  a  crystalline 
crust.     Examine  a  portion  of  this  microscopically  and  observe  the 
feathery  masses  and  sheaves  of  fine  white  needles,  characteristic  of 
tyrosine.     Filter  this  off   and  evaporate    the  filtrate    still  further. 
Leucine  separates  out  on  standing,  and,  examined  microscopically, 
shows  rounded  cones  writh  a  radiating  striation.     Make  a  drawing 
of  the  crystals  of  tyrosine  and  leucine. 


CHAPTER  VI. 

THE    COAGULATION    OF    BLOOD. 
Factors  concerned. 

1.  Fibrinogen  (Fgn.)  a    globulin,  present   in    blood- 
plasma.    It  is  soluble  in  dilute  salt  solutions,  acids  and 
alkalies,  insoluble  in   distilled  water.      It  coagulates  at 
57°  C.     It  is  precipitated  by  half-saturation  with  sodium 
chloride. 

2.  Pro-throinbin  (P)  a  substance  of  unknown  composi- 
tion, found  in  plasma,  attached  to  the  fibrinogen.     It  is 
destroyed  by  boiling. 

3.  Thrombokinase  (K)  a  substance  found  in  all  tissues 
and  also  liberated  in  the  blood  by  the  disintegration  of 
leucocytes  and  blood-platelets.     It  converts  pro-thrombin 
into  thrombin,  under  certain  conditions. 

4.  Calcium  salts,  found  in  plasma,  and  necessary  for 
the  action  of  thrombokinase.     The  calcium  salts  must  be 
of  such  a  nature  that  they  are  ionised  in  solution. 

5.  Thrombin  (T),  a  ferment  formed  by  the  interaction 
of  2,  3  and  4.     It  probably  splits  fibrinogen  into  serum- 
globulin  and  fibrin.     The  latter,   being  insoluble  in  the 
constituents  of  normal  plasma,  comes  out  of  solution  and 
with  the  corpuscles  forms  the  clot. 


100 


THE    COAGULATION     OF    BLOOD. 


[CH.   VI. 


The  phenomena  of  blood  coagulation  are  represented 
in  the  following  scheme  : — 


Blood  Plasma. 


Tissues, 

supply 


Water 

Salts 

Albumin 


Fgn. 


Corpuscles. 

White.     Platelets.     Red. 
liberate 


Serum-globulin  +  Fibrin 


Serum. 


Clot. 


Coagulation  is  hindered  by 

1.  Cooling. 

2.  Substances  which  precipitate    calcium   salts,    or 
convert   the   calcium  into  the  non-ionised   condition,  as 
oxalates,  citrates  and  soap  solutions. 

3.  Alkalies,  which  prevent  the  liberation  of  K  by  the 
corpuscles,  delay  the  action  of  T,  and  tend  to  dissolve 
fibrin. 

4.  Strong  salt  solutions,  which  have  a  similar  action. 

5.  Anti-thrombin,    a     substance     found     in     smal] 
amounts  in  the  plasma,  and  in  relatively  large  amounts 
in  extracts  of  the  head  of  the  leech.      It  combines  with  T 
to  render  it  inactive. 

6.  Anti-kinase,   found  in   the   blood,  after  the  slow 
injection  into  the  blood  stream  of  certain  substances,  as 
tissue-extracts,  certain  snake-venoms,  etc. 

7.  Fluorides,  which  precipitate  calcium  salts  and  pre- 
vent the  liberation  of  K. 


CH.    VI.]  THE    COAGULATION  ,  QF    BLOQT).  101 

Preparation  of  fibrin  ferment  (thrombin).  Blood  serum  is  treated  with 
four  or  five  times  its  volume  of  strong  alcohol,  well  stirred  and  allowed  to 
stand  for  two  or  three  days.  The  precipitate  is  collected,  dried  on  filter 
paper  in  the  air,  and  extracted  with  water.  The  filtered  extract  contains 
fibrin  ferment. 

Preparation  of  "  salted  "  plasma.  Two  litres  of  water  are  placed  in  a 
large  bottle  or  jar  (provided  with  a  tightly-fitting  stopper)  and  the  level  of 
the  fluid  marked  by  a  label.  The  water  is  poured  off  and  400  c.c.  of  a 
saturated  solution  of  magnesium  sulphate  substituted.  Blood  is  collected 
in  the  bottle  till  the  level  is  reached,  care  being  taken  to  ensure  thorough 
mixing  with  the  salt  solution  by  stopping  the  flow  of  blood  from  time  to 
time  and  turning  the  bottle  upside  down.  The  corpuscles  are  removed  by 
centrifugalisation  and  the  plasma  pipetted  off.  It  should  be  kept  in  a 
refrigerator  till  required. 

192.  The  clotting  of  salted  plasma.     Take  2  c.c.  of  salted 
plasma  in  a  test  tube,  add   10  c.c.   of  water,  and  divide  into  two 
portions,  A  and  B.     To  A  add  a  few  drops  of  fibrin  ferment  (or  of 
serum).     Place  both  tubes  in  the  warm  bath  at  40°  C.  and  examine 
from  time  to  time.     Clotting  takes  place  in  both  tubes,  but  much 
more  rapidly  in  A  than  in  B. 

NOTE. — Dilution  with  water  decreases  the  concentration  of  the  magnesium 
sulphate,  so  that  any  fibrin  formed  by  the  ferment  (which  can  now  act  on  the 
fibrinogen)  becomes  insoluble  in  this  low  concentration  of  salt. 

193.  The  preparation  of  fibrinogen.    To  20  c.c.  of  the  salted 
plasma    add    an    equal    volume  of  a  saturated  solution  of  sodium 
chloride.     A  precipitate  of  fibrinogen  is  formed.     Allow  the  tube  to 
stand    for    a    few  minutes  and  then  filter  through  a   small  paper. 
Scrape  the  precipitate  off  the  paper  and  treat  it  with  about  5  c.c.  of 
5  per  cent.  NaCl.     The  fibrinogen  dissolves. 

NOTE.— If  bird's  blood  be  drawn  directly  into  a  clean  vessel  in  such  a  way 
that  contact  with  the  tissues  is  absolutely  avoided,  it  clots  very  slowly.  This  is 
because  the  leucocytes  are  very  stable  and  do  not  liberate  thrombokinase.  If 
this  blood  be  centrifugalised  at  once,  a  non-clotting  plasma  is  obtained. 
Fibrinogen  can  readily  be  prepared  from  this  by  the  method  given  in  Ex.  24. 
The^ suspension  so  obtained  is  dissolved  in  dilute  salt  solution. 

194.  Divide  the  solution  thus  obtained  into  two  portions,  C 
and  D.     To  C  add  two  drops  of  fibrin  ferment.     Place  both  tubes 
in  the  warm  bath  and  observe  them  at  intervals.     C  clots  rapidly  ; 
D  very  slowly. 


10^  THE     COAGULATION     OF    BLOOD.  [CH.    VI. 

195.  The  heat-coagulation  of  fibrinogen.     Heat  5  c.c.  of 
salted  plasma  as  in  Ex.   10.     Notice  the  coagulation  of  fibrinogen 
which  occurs  at  56°  C.     Continue  heating  to  60°  C.  and  then  filter. 
Dilute  the  filtrate  as  in  Ex.  193  ;  add  fibrin  ferment,  and  place  on 
the  warm  bath.     Coagulation  does  not  occur. 

Preparation  of  oxalate  plasma.  Blood  is  drawn  as  in  the  prepar- 
ation of  salted  plasma  into  a  bottle  which  has  200  c.c.  of  a  1  per  cent, 
solution  of  potassium  oxalate  in  place  of  the  400  c.c.  of  saturated 
magnesium  sulphate.  The  plasma  is  separated,  as  before,  by  centrifugali- 
sation. 

196.  The  clotting  of  oxalate  plasma.     Dilute  5  c.c.  of  the 
plasma  with  10  c.c.  of  distilled  water  and  divide  into  three  portions 
E,    F,    and  G.     To   E  add  a  few  drops  of    1   per  cent,  calcium 
chloride ;  to  F,  a  few  drops  of  fibrin  ferment  or  serum.     Place  the 
three  tubes  on  the  water  bath  and  observe  them  at  intervals.     E 
clots  in  a  few  minutes ;  F  clots  slowly  ;  G  does  not  clot. 

Preparation  of  fluoride  plasma.  This  is  prepared  as  oxalate  plasma 
using  a  3  per  cent,  solution  of  sodium  fluoride  in  place  of  the  1  per  cent, 
potassium  oxalate. 

197.  The  clotting  of  fluoride  plasma.    Dilute  5  c.c.  with  10 
c.c.  of  water  and  divide  into  three  portions,  H,  K,  and  L.    To  H  add  a 
few  drops  of  1  per  cent,  calcium  chloride  ;  to  K  a  few  drops  of  fibrin 
ferment.     Place   the    three    tubes    in    the  warm -bath  and  observe 
them  at  intervals.     K  clots  rapidly ;  H  and  L  do  not  clot. 


CHAPTER  VII. 

THE  RED  BLOOD  CORPUSCLES  AND  THE 
BLOOD  PIGMENTS. 

A.     The  Laking  of  Blood. 

The  red  corpuscles  consist  of  an  envelope  and 
meshwork  called  the  stronia,  which  encloses  a  solution 
of  haemoglobin  and  various  salts.  The  stroma  con- 
sists of  a  protein,  probably  a  histone,  with  which  is 
associated  a  lipoid  material,  related  to  cholesterin  and 
lecithin.  The  envelope  behaves  as  a  semi-permeable 
membrane  to  a  great  many  solutions,  readily  allowing 
water  to  pass  into  or  from  the  corpuscle,  but  pre- 
venting the  passage  of  most  salts  and  other  dis- 
solved substances.  Thus  if  the  corpuscles  are  placed 
in  a  solution  which  has  a  higher  osmotic  pressure  than 
the  fluid  within  the  corpuscles,  water  passes  out  of  the 
corpuscle,  which  therefore  shrinks.  Such  fluids  are  called 
"  hypertoiiic."  If  they  be  placed  in  fluids  of  a  lower 
osmotic  pressure  ("  hypotonic "),  water  passes  into  the 
corpuscle  to  equalise  the  pressures,  but  salts  cannot  pass 
out.  The  corpuscles  swell  and  the  expansion  may  be 
sufficient  to  lead  to  the  disruption  of  the  envelope,  so  that 
the  enclosed  haemoglobin  passes  into  the  body  of  the 
solution.  This  bursting  of  the  corpuscles  is  known  as 
laking  or  haemolysis.  A  solution  of  the  same  osmotic 
pressure  as  that  of  the  fluid  within  the  corpuscle  is  said 
to  be  "isotonic"  or  "normal."  For  mammalian  blood 
0*9  per  cent,  sodium  chloride  is  normal ;  for  frog's  blood, 
O65  per.  cent.  Other  physical  means  of  inducing  hae- 
molysis are  by  repeatedly  freezing  and  thawing  the  blood, 


104  THE  RED  BLOOD  CORPUSCLES.        [CH.  VII. 

or  by  warming  to  60°  C.  The  envelope  can  also  be  ruptured 
by  chemical  means.  Certain  substances,  such  as  the  bile 
salts,  ether,  chloroform,  acids,  alkalies,  and  saponin  are 
solvents  for  the  lipoids. 

Another  method  of  inducing  haemolysis  is  by  the 
addition  of  certain  organic  substances  developed  in  certain 
animals.  Thus  rabbit's  corpuscles  that  have  been  washed 
with  isotonic  saline  are  laked  when  treated  with  the 
blood  serum  of  a  dog.  This  haemolytic  power  of  dog's 
serum  on  rabbit's  blood  is  very  much  increased  by 
previously  injecting  the  dog  with  rabbit's  blood. 

198.  Have  two   burettes,  one  containing  1   per  cent,  sodium 
chloride,  the  other  distilled  water. 

Label  a  series  of  clean,  dry  test-tubes,  A,  B,  C,  etc., 

In  A  place  4-5  c.c.  NaCl  and  5'5  c.c.  H2O  =  45%  NaCl. 

-D        ,,          J  ,,  -J  ,,  -     J      ,, 

Cz.c  4_.c  —  .^^ 

,,  J   J  ,,  T   J  ,,  w>~>  ,, 

D     „  "  6  „  4  „      =  -6    „ 

E     „      6-5  „  3'5  ,,      =  "65  „ 

F     ,,      7  ,,  3  ,,       =  *7    ,, 

To  each  tube  add  three  drops  of  freshly  defribinated  blood, 
mix  by  inverting  and  allow  the  tubes  to  stand  for  a  few  minutes. 
A  will  be  translucent,  the  corpuscles  being  fully  laked.  F  will  be 
opaque.  Note  the  dilution  which  just  causes  laking.  It  is. 
generally  about  '55  per  cent. 

NOTE. — The  solution  that  just  causes  laking  is  hypotonic  to  the  blood, 
indicating  that  the  corpuscles  can  absorb  a  considerable  quantity  of  fluid  before 
the  envelope  is  ruptured. 

199.  To  5  c.c.  of  0'9  per  cent,  sodium  chloride  add  some  ether 
and    shake    vigorously.     Then    add  three   drops  of  blood,  mix  by 
inversion.     The  blood  is  laked. 

200.  To  a  -2  per  cent,  solution  of  bile  salts  in  normal  saline 
add  three  drops  of  blood.     It  is  laked. 


CH.    VII.]  HAEMOGLOBIN.  105 

201.  Add  some  blood   to   a  2  per   cent,  solution  of   urea  in 
water.     The  blood  is  laked. 

202.  Repeat   the  experiment  with  a  2   per   cent,   solution  of 
urea  in  normal  saline.     The  blood  is  not  laked. 

B.    Haemoglobin  and  its  Derivatives. 

Haemoglobin  (Hb)  is  a  compound  protein,  being  a 
member  of  the  group  of  chromoproteiiis.  It  is  formed  by 
the  union  of  a  pigmented  non-protein  substance  containing 
iron,  and  called  haematin  (Hn),  with  globin,  a  member 
of  the  histone  group  of  proteins. 

It  is  soluble  in  water  and  dilute  salt  solutions: 
insoluble  in  ether  and  alcohol. 

It  is  decomposed  by  acids  and  alkalies  into  haematin 
and  globin.  It  is  decomposed  and  coagulated  by  heat. 

It  forms  compounds  with  oxygen  and  carbon  mon- 
oxide, called  oxyhaemoglobin  (Hb-O2)  and  carboxyhae- 
moglobin  (Hb-CO).  Both  are  dissociated  into  Hb  and  the 
gas  by  exposure  to  a  vacuum.  Hb-CO  is  much  more 
stable  than  Hb-O2,  and  the  avidity  of  Hb  for  CO  is  more 
than  130  times  greater  than  the  avidity  of  Hb  for  O2. 
A  small  percentage  of  CO  in  the  air  breathed  will  thus 
result  in  the  formation  of  relatively  considerable  amounts 
of  Hb-CO  in  the  blood.  This  can  be  converted  into 
Hb-O2  by  exposure  to  a  high  tension  of  O2,  such  as  is 
obtained  by  breathing  pure  O2. 

The  Hb-O2  obtained  from  certain  animals  crystallises 
readily,  but  the  crystals  differ  somewhat,  according  to  the 
animal  from  which  they  are  obtained.  Also  the  volume 
of  O2  combining  with  1  gram  of  Hb  varies,  the  figure  for 
the  horse  being  1'34  c.c.  of  O2  per  gram  of  Hb.  The 
oxygen  is  probably  united  to  the  iron  of  the  haematin 
molecule,  the  reaction  Fe  +  Q9<  >FeO9  being  the  basis  of 
the  reaction  Hb  +  O2<— ~rHb-O2. 


106 


THE     RED     BLOOD    CORPUSCLES. 


[CH.   VII. 


The  ratio 


volume  of  O9  evolved  in  c.c. 


is    called    the 


-401. 


weight  of  iron  in  grams, 
specific  oxygen  capacity. 
Theoretically  it  is 

O2     1  molecular  volume  O0    '22,394 
Fe~l    gram    molecule   Fe~  55*85 
Recent  analyses  of  the  blood  of  various  animals  have 
given  the  value  401*8,  which  agrees  very  closely  with  the 
theoretical. 

The   volume   of    oxygen   loosely   held   by   1   gram  of 
Hb,O2  is  1-345  c.c. 

So    the   minimum    molecular   weight   of   oxyhaemo- 

22  394 
globin  is  -          =  16,712. 


The  method  of  formation  of  certain  of  the  derivatives 
of  haemoglobin  can  be  represented  as  follows : — 

Haemoglobin. 

ft  Methaemoglobin. 


Ox  y  haemoglobin. 


Globin  +  Acid  haematin 


Globin  -f  Alkaline  haematin. 


v 

Haemochrom  oge  n . 


v 

Acid  haematoporphyrin  +  Iron, 


A Ikaline  haematoporpli  i/rin . 


CH.    VII.] 


THE     BLOOD     PIGMENTS. 


107 


203.     Crystallisation  of  oxy haemoglobin  (Rapid  method). 

To  a  few  c.c.  of  defibrinated  dog's  blood  in  a  test  tube  add  ether,  drop 
by  drop,  till  the  blood  is  completely  laked.  Add  to  the  blood  a  pinch 
of  finely  powdered  ammonium  oxalate ;  allow  the  salt  to  dissolve  by 
gentle  shaking,  and  let  the  tube  stand.  Crystals  of  oxyhaemoglobin 
separate  out,  especially  if  the  solution  is  cooled  to  0°C.  by  means  of 
ice.  Examine  them  microscopically,  and  note  that  they  are  in  the 
form  of  thin  rhombic  prisms. 

Make  a  drawing  of  the  crystals. 

NOTE. — This  experiment  does  not  always  succeed  as  described.  If  the 
blood  fails  to  crystallise  out  in  an  hour,  place  a  drop  on  a  slide,  spread  it  out  to 
form  a  thin  layer  and  leave  it  for  five  minutes  ;  cover  with  a  slip  and  note  the 
crystals  of  oxyhaemoglobin  that  form  at  the  edges. 

C.     The  Spectroscopic  Examination  of  the  Blood  Pigments. 
The  use  of  the  Direct-vision  Spectroscope. 

The  instrument  described  is  the  small  pocket  spectroscope,  with 
wave-length  scale  attached,  manufactured  by  Zeiss  and  Co.  The 
instrument  (fig.  2)  consists  of  two  tubes.  The 
shorter  tube  A  contains  a  transparent  photo- 
graphic scale  of  wave-lengths,  with  a  mirror  to 
project  its  image  into  the  field  of  vision.  By 
means  of  the  tube  D  this  scale  can  be  focussed 
and  by  the  screw  F  it  can  be  adjusted  to  its 
proper  position.  The  tube  G  contains  a  series  of 
alternating  prisms  of  crown  and  flint  glass, 
arranged  to  allow  the  spectrum  to  be  observed  by 
the  eye  in  the  line  of  the  tube.  The  tube  B  which 
slides  on  G  has  a  vertical  slit,  the  width  of 
which  can  be  adjusted  by  turning  the  collar  E. 

To  adjust  the  spectroscope  :  see  that  D  and  B 
are  pushed  in  as  far  as  they  will  go.  Look 
through  C  towards  the  light  with  A  to  your  left, 
and  turn  E  till  the  spectrum  is  only  just  visible. 
(It  is  most  important  to  use  an  extremely  narrow  C 

'slit.)  Slide  B  out  very  slowly  (in  most  instru  Fig.-  2. — Zciss'  dircct- 
ments  for  3|  divisions  as  marked  on  the  barrel  G)  vision  spectroscope 
till  fine  black  vertical  lines  can  be  seen  in  the 
spectrum,  and  notice  particularly  a  fine  black  line 
immediately  to  the  left  of  the  narrow  strip  of  yellow.  This  line  is  known 
as  the  D  line  of  Fraunhofer.  The  wave-length  of  it  is  -59/*,  a  position 
indicated  on  the  scale  by  the  division  marking  it  (the  one  to  the  right  of 
0-6)  being  produced  further  down  than  any  other.  If  necessary  alter  the 


B 


with 
scale 


wave-length 


108  THE  RED  BLOOD  CORPUSCLES.        [CH.  VII. 

position  of  the  scale  by  turning  the  screw  F  until  the  D  line  exactly 
coincides  with  the  division  mentioned.  If  the  instrument  has  to  be 
adjusted  at  night-time,  when  the  D  line  cannot  be  observed,  set  the  scale 
by  use  of  the  emission-spectrum  of  sodium  (obtained  by  placing  a  few 
crystals  of  common  salt  on  the  wick  of  a  spirit  lamp).  The  emission 
spectrum  of  sodium  exactly  corresponds  to  the  D  line.  The  scale  is  so 
drawn  that,  if  it  be  set  in  position  as  described,  the  wave-  length  of  light 
in  any  part  of  the  visible  spectrum  can  be  read  directly. 

The  numbers  on  the  scale  indicate  wave-lengths  in  thousandths  of  a 
millimetre,  the  unit  being  I/A.  In  the  more  recent  patterns  the  wave- 
lengths are  given  in  millionths  of  a  millimetre,  the  unit  being  IX.  Thus 
the  wave-length  of  the  D  line  is  589X.  The  other  Fraunhofer  lines  that 
can  be  readily  observed  with  the  instrument  are  C(657X),  E  (527X), 
b  (518A)  and  F  (486  X). 

To  observe  absorption  spectra :  slightly  open  the  slit  of  the  spec- 
troscope, thus  obtaining  a  better  illumination.  Direct  the  instrument  to 
the  light,  and  place  the  test  tube  containing  the  fluid  to  be  examined 
directly  in  front  of,  and  touching,  the  tube  B,  with  its  axis  'parallel  to  the 
slit,  taking  care  not  to  interfere  with  the  illumination  of  the  scale.  With 
strong  solutions  of  certain  pigments  observed  in  this  way  it  is  often 
difficult  to  avoid  illuminating  the  two  ends  of  the  spectrum,  the  light  being 
reflected  from  the  sides  of  the  tubes,  and  not  passing  through  the  solution. 
To  obviate  this  it  is  perhaps  better  to  place  the  solution  in  a  beaker, 
remembering  that  the  absorption  of  light  increases  with  the  depth  of 
layer  examined,  as  well  as  with  the  concentration  of  the  pigment.  For 
accurate  work  the  haematoscope  should  be  employed.  This  is  a  vessel 
with  parallel  glass  slides  1  cm.  apart. 

In  handling  the  instrument  the  screw  F  is  very  liable  to  be  turned  and 
so  the  position  of  the  scale  to  be  shifted.  From  time  to  time,  therefore, 
the  slit  should  be  narrowed,  and  an  observation  made  to  ascertain  whether 
any  shifting  of  the  scale  in  reference  to  the  D  line  has  occurred. 

Record  the  absorption  of  light  of  the  various  pigment  solutions  on  the 
blank  scale,  to  be  found  towards  the  end  of  the  book.  Fill  in  with  black 
pencil  marks  the  exact  parts  of  the  spectrum  where  light  is  absorbed, 
leaving  the  remainder  blank.  It  will  not  be  found  advisable  to  use 
coloured  pencils. 

204.  Oxyhaemoglobin.  Take  5  c.c.  of  distilled  water  in  a  test 
tube,  and  add  one  drop  of  denbrinated  blood,  shake  well  and  observe 
the  spectrum  of  dilute  oxyhaemoglobin.  There  are  two  absorption 
bands  in  the  green.  The  one  near  the  D  line  (the  a  band)  is  some- 
what narrower  and  darker  than  the  ft  band.  The  middle  of  a  is 
about  X  578,  and  that  of  ft  about  X  540. 


CH.    VII.]  HAEMOGLOBIN  109 

205.  Add  two  more  drops  of  defibrinated  blood  and  examine 
again.     The  spectrum  has  become  very  much  cut  off,  especially  at 
the  blue  end :  the  absorption  bands  have  probably  merged  into  one, 
leaving  a  little  patch  of  blue  light  and  a  broader  belt  of  red  light  on 
the  two  sides.     Record  the  spectrum  of  the  solution  on  the  chart  as 
that  of  a  medium  solution  of  oxyhaemoglobin. 

206.  Add  another  drop  or  two  of  defibrinated  blood,  and  note 
that  the  blue  light  becomes  absorbed,  light  only  coming  through  in 
the    red.     (Strong    solution.)       If  the  concentration  is   still  further 
increased,  the  red  also  is  absorbed. 

NOTE. — It  is  important  to  observe  that  a  medium  solution  of  oxyhaemo- 
globin has  a  single  band  in  the  green. 

207.  Haemoglobin  (reduced  haemoglobin).    Treat  5  c.c.  of 
water  with  one  drop  of  defibrinated  blood  and  thus  obtain  a  solution 
of  oxyhaemoglobin  of  such  a  strength  that  two  well-marked  absorp- 
tion  bands  can   be    observed.     Add    two    drops    of    a    solution  of 
ammonium  sulphide,  mix  and  warm  to  about  50° C.,  avoiding  any 
unnecessary  shaking :  or  if  Stokes'  fluid  is  obtainable,  add  two  or 
three  drops,  in  which  case  there  is  no  necessity  to  warm.     Note,  in 
the  latter  case,  that  the   bright  scarlet  colour  of   oxyhaemoglobin 
gives    place    to    the    less    vivid    colour    of    reduced    haemoglobin. 
Examine  the  solution  spectroscopically.     There  is  a  single  broad 
band  in  the  green  which  overlaps  the  space  enclosed  by  the  two 
bands  of  oxyhaemoglobin,  and  is  fainter  than  either.     Its  centre  is 
.about  X  565. 

NOTES. — Stokes'  fluid  is  prepared  as  follows  :  dissolve  3  grams,  of  ferrous 
sulphate  in  cold  water  :  add  a  cold  aqueous  solution  of  2  grams,  of  tartaric  acid 
and  make  the  solution  up  to  100  c.c.  with  water.  Immediately  before  use  add 
strong  ammonia  until  the  precipitate  first  produced  is  redissolved.  It  rapidly 
absorbs  atmospheric  oxygen  and  must,  therefore,  be  freshly  prepared.  Its 
great  advantage  over  ammonium  sulphide  is  that  it  can  be  used  in  the  cold, 
whilst  with  the  sulphide  the  solution  must  be  warmed. 

208.  Place  your  thumb  over  the  top  of  the  test  tube  contain- 
ing the  reduced  haemoglobin  and  shake  vigorously.  Examine 
immediately  with  the  spectroscope,  and  note  that  the  two  bands  of 
.oxyhaemoglobin  have  reappeared  owing  to  the  oxidation  of  the 


110  THE  RED  BLOOD  CORPUSCLES.        [CH.  VII. 

haemoglobin  by  the  oxygen  of  the  air.  If  the  tube  be  allowed  to 
stand  for  a  short  while,  reduction  may  appear  again  from  excess  of 
reducing  reagent  present. 

209.  Carboxyhaemoglobin.     Obtain  some  CO-haemoglobin 
that  has  been  prepared  by  passing  a  stream  of  carbon  monoxide  or 
coal-gas  through  a  solution  of  oxyhaemoglobin.     Notice  the  peculiar 
bluish  tinge  of  the  solution.     Examine  a  portion  spectroscopically , 
and,  if  necessary,  add  water  till  two  well-marked  bands  are  visible. 
Note  that  they  are  very  similar  to  the  two  bands  of  oxyhaemoglobin. 
Accurate    observation,    however,    will    show    that    they    are    both 
slightly   nearer  the  violet  end    of    the  spectrum,  the   middle  of  « 
being  X  572  and  of  ft  \  535.      . 

NOTES. — 1.  A  small  amount  of  ether  added  to  the  blood  facilitates  the 
preparation  of  Hb-CO  in  preventing  excessive  frothing. 

2.  If  the  student  can  satisfy  himself  of  the  difference  between  the  position 
of  the  absorption  bands  of  Hb-C>2  and  Hb-CO,  he  can  always  obtain  a 
sample  of  Hb-O2  for  comparison  with  an  unknown  solution  by  pricking  his 
finger. 

210.  Take  a  portion  of  the  diluted  solution  of  CO-haemoglobin 
just  examined,  treat  it  with  a  few  drops  of  ammonium  sulphide, 
warm  in  a  bath  at  50°  C.  for  three  minutes  and  examine  with  the 
spectroscope.     No  change  takes  place  in  the  spectrum.     (Distinction 
from  oxyhaemoglobin.) 

211.  In  two  test  tubes    place  2    or    3    c.c.    of    solutions    of 
oxyhaemoglobin  and  CO-haemoglobin  of  the  same  depth  of  colour. 
Fill  the  test  tubes  with  water  and  mix  well.     Note  that  the  CO- 
haemoglobin    takes    on   a    well-marked    carmine    tint,    whilst    the 
oxyhaemoglobin  turns  yellow.     This  simple  test,  which  can  only  be 
seen   on  extreme    dilution,    rapidly  serves  to   distinguish    the  two 
compounds. 

212.  Katyama's    test    for     CO-Haemoglobin    in    blood. 

Add  5  drops  of  blood  to  10  c.c.  of  water  :  then  add  5  drops  of 
orange-coloured  ammonium  sulphide.  Mix  and  add  enough  strong 
acetic  acid  to  make  the  mixture  faintly  acid.  With  blood  containing 


CH.    VII.]  METHAEMOGLOBIN.  Ill 

CO,  a  rose-red  colour  appears :  with  normal  blood  a  dirty  greenish- 
grey.  The  colour  is  still  perceptible  with  one  part  of  the  CO-blood 
to  5  of  normal  blood. 

213.  Methaemoglobin.     To  5  c.c.  of  water  add  four  drops 
of  defibrinated  blood.     To  the  strong  solution  of  oxyhaemoglobin 
thus  formed  add  two  drops  of  a   saturated  solution  of  potassium 
ferricyanide.     The  colour  of  the  solution  changes  to  a  chocolate- 
brown.     Examine  with  the  spectroscope :  there  is  visible  a  promi- 
nent band  in  the  red,  with  its  centre  at  about  X  630.     There  is 
marked  absorption  of  the  blue  end  of  the  spectrum.     Dilute  with 
an  equal  bulk  of  water  and  examine  again  :  two  faint  bands  appear 
in  the  green  in  the  position  of  the  bands  of  oxyhaemoglobin. 

213A.  Dilute  the  solution  of  methaemoglobin  thus  obtained 
with  another  volume  of  water.  Treat  5  c.c.  of  this  with  two  or 
three  drops  of  ammonium  sulphide  and  examine  immediately.  The 
colour  changes  to  a  red :  the  absorption  band  in  the  red  disappears, 
and  the  spectrum  of  oxyhaemoglobin  is  seen.  Warm  the  solution 
to  50°  C.  and  allow  it  to  stand  for  a  short  time  (possibly  with  the 
addition  of  another  drop  or  two  of  the  reducing  reagent).  The  two 
bands  give  place  to  the  single  band  of  reduced  haemoglobin.  Shake 
with  air  :  oxyhaemoglobin  is  reformed. 

214.  Take  a  few  c.c.  of  defibrinated  blood  in  a  test  tube,  add 
an  equal  quantity  of  water  and  warm  to  50° C.  to  lake  the  blood. 
To  the   solution   thus    obtained  add    an    equal    bulk    of    saturated 
potassium  ferricyanide.     Mix  by  giving  one  shake,  and  then  hold 
the  tube  at  rest  in  an  oblique  position  for  a  short  time.     Note  the 
bubbles  of  gas  (oxygen)  that  are  evolved. 

NOTES. — 1.  Oxyhaemoglobin  is  converted  into  methaemoglobin  by  the 
action  of  oxidising  reagents,  such  as  ferricyanides,  nitrites,  chlorates,  and 
permanganates,  and  in  the  body,  by  the  action  of  many  aromatic  substances, 
such  as  phenol. 

2.  The  reaction  is  peculiar  in  that  an  amount  of  oxygen  is  evolved 
equivalent  to  that  held  in  combination  by  the  oxyhaemoglobin,  although 
methaemcglobin  contains  the  same  percentage  of  oxygen  as  oxyhaemoglobin. 
This  reaction  is  the  basis  of  the  modern  method  of  estimating  the  amount  of 
oxygen  in  the  blood. 


112  THE  RED  BLOOD  CORPUSCLES.        [CH.  VII. 

The  reaction  might  be  represented  by  the  following  equation  : — 

/O  ..O 

Hb\    |  +          02        =  Hb'^  +         02 

O  \0 

Oxyhaemoglobin.  From  ferricyanide.         Methaemogiobin.  Gas. 

The  oxygen  is  represented  as  being  in  a  different  state  of  combination  in 
methaemogiobin,  since  it  cannot  be  removed  by  submitting  the  compound  to  a 
vacuum. 

3.  When  methaemogiobin  is  treated  with  a  reducing  reagent,  the  first 
change  that  occurs  is  that  the  linkage  of  the  oxygen  atoms  reverts  to  that  oi 
oxyhaemoglobin  ;  later  the  oxygen  is  removed  and  reduced  haemoglobin 
formed. 

215.  Acid  haematin.     To  5  c.c.  of  water  add  four  drops  of 
defibrinated  blood  and  five  drops  of  strong  acetic  acid  and   heat. 
The  colour  changes  to  brown  ;  and  the  solution  showTs  an  absorption 
band  in  the  red,  which   is  further  from  the   D   line   than   that  oi 
methaemogiobin.     Its  centre  is  about  X  650. 

216.  Acid  haematin  in  ethereal  solution.    Treat  a  few  c.c 
of  defibrinated  blood  with  one  drop  of  strong  hydrochloric  acid  and 
a  few  c.c.  of  acetic  acid :  extract  this  with  about  5  c.c.  of  ether  b\ 
gentle  shaking,  pour  the  ether  into  a  clean  tube  and  examine  it  with 
the  spectroscope.     There  is  a  prominent  band  in  the  red  (centre  ^ 
638) ;  on  dilution  with  ether  three  other  bands  can  be  seen ;  a  ver\ 
narrow  one  with  centre  X  582 ;  a  broad  one  stretching  from  aboui 
X  555  to  X  530  and  another  from  X  512  to  X  498. 

217.  Alkaline  haematin.    Treat  a  moderately  strong  solutior 
of  oxyhaemoglobin  with  a  few  drops  of  strong  sodium  hydroxide 
and   warm.     The   colour    changes    to    brown.     Examine  with  the 
spectroscope :  a  faint  band  is  seen  in  the  red,  stretching  from  the 
D  line  to  about  X  630.     There  is  a  considerable  absorption  of  the 
blue  end  of  the  spectrum. 

NOTE. — A  band  of  alkaline  methaemogiobin  in  the  yellow-green  is  oftei 
seen  as  well. 

218.  Alkaline    haematin    in    alcohol.      Mix    defibrinatec 
blood  into  a  thin  paste  with  solid  potassium  carbonate  and  evaporat< 
to    complete  dryness  on  a  water  bath.     Powder  the  residue,  boi 


CH.    VII.]  DERIVATIVES     OF     HAEMOGLOBIN.  113 

with  alcohol  in  a  flask  on  the  water-bath  and  filter.  The  solution 
contains  alkaline  haematin  free  from  proteins.  It  shows  the 
absorption  band  of  alkaline  haematin  more  distinctly  than  the  crude 
aqueous  solution  prepared  in  Ex.  217. 

219.  Haemochromogen  (reduced  alkaline  haematin).     Pre- 
pare a  solution  of  alkaline  haematin  from,  dilute  oxyhaemoglobin 
as  in  Ex.  217.     Treat  it  with  a  few  drops  of  ammonium  sulphide. 
The   colour  of   the   solution   changes  to    red.     Examine    with   the 
spectroscope.     Two  absorption  bands  are  seen  in  the  green.     The 
band  nearer  the  D  line  ( the  a  band)  is  very  prominent  and  sharply 
defined,    with    its    centre  at  about  X  558.     The  /3  band  is   much 
fainter  and  has  its  centre  at  X  520. 

NOTE. — In  very  dilute  solutions  only  the  a  band  can  be  seen.  The 
absorption  of  light  in  this  region  is  so  intense  that  if  a  solution  of  oxyhaemoglobin , 
so  dilute  that  its  absorption  bands  cannot  be  readily  seen,  be  converted  by 
appropriate  means  into  haemochromogen,  the  a  band  of  this  pigment  is  usually 
observable. 

220.  Acid  haematoporphyrin.    To  a  few  c.c.  of  concentrated 
sulphuric  acid  in  a  test  tube  add  two  drops  of  defibrinated  blood 
(see  note  to  Ex.  221)  and  mix  by  gentle  shaking.     Note  the  rich 
purple    colour    of    the    solution.     Examine    with  the  spectroscope. 
Two  bands  are  seen  :  the  a  band,  with  centre  at  X  600,  being  fainter 
and  narrower  than  the  ,8  band,  centre  X  554. 

221.  Alkaline  haematoporphyrin.    To  the  solution  of  acid 
haematoporphyrin    just    prepared    add    five  or  six   more   drops  of 
defibrinated  blood,  shaking  gently  after  the  addition  of  each  drop. 
Pour  the  strong  solution  into  about  50  c.c.  of  cold  water  in  a  beaker, 
stir  well  and  note  the  precipitate  that  rises  to  the  surface.     Transfer 
this  precipitate  to  a  test  tube  by  means  of  a  rod ;  treat  it  with  a  few 
c.c.  of  alcohol  and  boil.     And  5  c.c.  of  sodium  hydrate.     A  solution 
of    alkaline    haematoporphyrin    is   thus    obtained,  which  examined 
spectroscopically    after    suitable    dilutions    shows    a  four   banded 
spectrum.    The  centres  of  the  bands  are  at  X  622,  X  576,  X  539  and 

A  504  approximately. 

i 


114  THE  RED  BLOOD  CORPUSCLES.        [CH.  VII. 

NOTE. — The  conversion  of  blood  pigment  into  haematoporphyrin  involves 
two  processes.  Firstly,  the  acid  splits  off  the  protein  constituent  (globin)  and 
forms  acid  haematin  ;  secondly,  the  acid  haematin  loses  its  iron  and  becomes 
haematoporphyrin.  The  first  change  is  effected  very  readily  even  by  dilute 
acids,  but  the  separation  of  the  iron  from  the  haematin  normally  requires  highly 
concentrated  mineral  acids.  It  has,  however,  been  shown  that  if  the  blood  be 
first  reduced  the  iron  is  split  off  with  much  greater  ease  by  the  acid.  An  efficient 
method  of  reducing  defibrinated  blood  is  that  of  "auto-reduction,"  in  which  a 
tightly  corked  vessel  full  of  blood  is  allowed  to  stand  for  a  few  days.  If 
exercises  220  and  221  be  carried  out  with  this  reduced  blood,  care  being  taken 
by  use  of  a  pipette  to  prevent  re-oxidation,  the  haemoglobin  is  entirely  converted 
into  haematoporphyrin,  no  trace  of  the  brown  haematin  being  left. 

222.  Preparation  of  haemin  crystals.  (Teichmann's 
crystals.)  Place  a  drop  of  defibrinated  blood  on  a  glass  slide,  add 
a  speck  of  sodium  chloride  and  rub  with  a  glass  rod  till 
the  salt  has  dissolved.  Evaporate  to  complete  dryness  by  support- 
ing the  slide  about  a  foot  above  a  small  flame.  >Rub  the  red 
residue  to  a  fine  powder  with  a  pen-knife,  collect  it  into  a  little  heap 
and  add  a  drop  of  glacial  acetic  acid  on  the  end  of  a  glass  rod.  Rub 
into  a  paste,  and  place  a  little  of  this  on  a  clean  slide,  add  a  drop  of 
glacial  acetic  acid,  cover  with  a  slip  and  cautiously  heat  over  a 
small  flame  till  it  just  boils.  Let  a  drop  more  of  the  acid  run  under 
the  slip  and  then  allow  to  cool.  Examine  microscopically  for  the 
brown  rhombic  prisms  of  haemin  (haematin  hydrochloride).  Draw 
them  in  the  space  provided  at  the  end  of  the  book. 

NOTES. — Care  must  be  taken  to  add  only  a  trace  of  sodium  chloride,  to 
dry  slowly,  and  to  observe  that  the  acid  mixture  really  boils  and  that  all  the 
acid  is  not  evaporated  off. 

This  test  can  be  applied  to  dilute  solutions  of  haemoglobin  by  acidifying 
with  acetic  acid,  precipitating  with  freshly  prepared  tannic  acid,  and  treating  the 
dried  precipitate  on  a  slide  with  a  trace  of  salt  and  glacial  acetic  acid  as  described 
above.  Suspected  blood  stains  on  linen,  instruments,  etc.,  should  be  extracted 
with  a  little  alkali,  the  solution  evaporated  to  dryness  and  treated  as  above. 


CHAPTER    VIII. 
THE    CONSTITUENTS    OB'    BILE. 

Bile  is  secreted  continuously  into  the  hepatic  ducts 
by  the  liver.  During  the  intervals  of  digestion  it  is  stored 
in  the  gall  bladder,  being  poured  into  the  duodenum  when 
the  acid  chyme  passes  through  the  pylorus. 

During  its  stay  in  the  gall  bladder  there  is  an  absorp- 
tion of  water  and  an  increase  in  the  protein  content, 
resulting  in  an  increase  in  the  specific  gravity  from  about 
1010  to  1040. 

The  percentage  composition  of  human  bile  varies 
considerably.  The  following  are  average  figures  : — 


From 
Gall  Bladder. 


Water  86 

Solids  14 

Bile  salts        9 

Protein | 

Bile  pigments) 

Cholesterin    ...         ...  0*2 

Lecithin  and  fat      ...  1/0 

Inorganic  salts         ...  0*8 


From 
Fistula. 

...     98 

...      2 

.  0-8 


...  0-06 

...  0-04 

.  0-8 


The  bile  salts  are  the  sodium  salts  of  glycocholic 
and  taurocholic  acids.  They  are  formed  by  the  condensa- 
tion of  cholalic  acid  (C24H40O5)  with  glycine  (amino-acetic 
acid,  CH2.NH2.COOH)  and  taurine  respectively.  Glycine 
is  one  of  the  products  obtained  by  the  hydrolysis  of 
proteins. 


116  THE    CONSTITUENTS     OF     BILE.  [cH.    VIII. 

Taurine  is  derived  from  a  similar  product,  cysteine. 
CH2SH  CH2.S03H 

CH.NH2       >       CH.2.NH2 

COOH 

Cysteine.  Taurine. 

The  bile  acids  are  hydrolysed  into  their  constituents 
by  boiling  acids  and  also  by  the  intestinal  bacteria. 

The    bile    salts   are    soluble    in   water    and    alcohol, 
insoluble  in  ether. 

Their  solutions  have  a  remarkably  low  surface  tension. 
(See  Hay's  test.) 

They  have  the  following  functions  : — 

1.  They  have  a  marked  adjuvant  action  on  pancreatic 
lipase.     (See  Ex.  116.) 

2.  They  are   solvents  for  the  fatty  acids  and  thus 
markedly  increase  the  absorption  of  fats.     (See  p.  65.) 

3.  They   thus  help   to   remove    the    fatty   film   sur- 
rounding the  protein,  and  allow  the  proteolytic  ferments 
to    act.     In    this    way,    by   assisting    the   absorption   of 
proteins,   they  diminish  bacterial  decomposition.     They 
are  not  direct  antiseptics. 

Preparation  of  Bile  Salts.— Mix  40  c.c.  of  ox  gall  with  enough  animal 
charcoal  (about  10  grams)  to  form  a  paste.  Evaporate  to  dryness  over  a 
water  bath,  stirring  at  intervals.  Grind  the  residue  in  a  mortar,  transfer 
it  to  a  flask,  add  about  70  c.c.  of  96  per  cent,  or  absolute  alcohol  and  boil 
on  the  water  bath  for  20  minutes.  Cool  and  filter  into  a  dry  beaker.  Add 
ether  to  the  filtrate  till  there  is  a  slight  permanent  cloudiness.  Cover 
the  beaker  with  a  glass  plate  and  allow  it  to  stand  in  a  cool  place  for  24 
hours.  A  crystalline  mass  of  bile  salts  separates  out.  The  crystals  are 
filtered  off  and  allowed  to  dry  in  the  air. 

For  the  following  tests  use  a  1  per  cent,  solution  of 
bile  salts  or  diluted  ox  or  sheep  gall :  — 

223.     Pettenkofer's   test  for  bile  salts.     To  5  c.e.  of  the 
solution  add  a  small   particle  of  cane-sugar  and  shake  or  warm 


CH.    VIII.]  THE     BILE     SALTS.  117 

till  this  has  completely  dissolved.  To  the  cooled  solution  add  5  c.c. 
of  concentrated  sulphuric  acid,  inclining  the  test  tube  so  that  the 
acid  settles  to  the  bottom.  Gently  shake  the  test  tube  from  side  to 
side.  As  the  fluids  gradually  mix  a  deep  purple  colour  develops. 

NOTES. — 1.  This  reaction  depends  on  the  production  of  furfurol  from  the 
cane-sugar  by  the  strong  acid.  (See  Ex.  76.) 

2:  If  too  much  cane-sugar  be  taken  the  fluid  will  turn  brown  or  black, 
owing  to  the  charring  produced. 

3.  Proteins  give  a  very  similar  reaction  with  furfurol  in  the  presence  of 
strong  acids.     Proteins  also  tend   to  give  a  brown  char  with  sulphuric  acid. 
For  these  reasons  it  is  advisable  to  remove  the  proteins   from  solution  before 
attempting  the  test. 

4.  The  purple  colour  obtained  is  only  stable  in  the  presence  of  strong 
sulphuric  acid.     It  disappears  on  dilution  with  water. 

5.  If  a  small  portion  of  the  coloured  fluid  be  diluted  with  50  per  cent, 
sulphuric  acid,  and  examined  with  the  spectroscope,  two  absorption  bands  will 
be  seen,  one  between  the  lines  C  and  D,  nearer  the  latter  ;    the  other  in  the 
green,  overlapping  E  and  B. 

6.  The  test  cannot  be  applied  directly  to  urine,  owing  to  the  presence  of 
chromogenic  substances  that  yield  intense  colours  with  sulphuric  acid. 

224.  Hay's  test  for  bile  salts.  Take  10  c.c.  of  the 
solution  in  a  test  tube.  Sprinkle  the  surface  with  flowers  of  sulphur 
and  note  that  they  fall  through  the  liquid  to  the  bottom  of  the  tube. 
Repeat  the  test  with  water,  noting  that  the  particles  remain  on  the 
surface. 

NOTES. — 1.  This  test  for  bile  salts  depends  on  the  remarkable  property 
that  they  possess  of  lowering  the  surface  tension  of  water,  thus  enabling  the 
particles  of  sulphur  to  sink  through  the  fluid. 

2.  The  test  is  of  great  value  for  the  detection  of  bile  salts  in  urine. 

3.  This  property  of   bile  salts  is  utilised  by  draughtsmen  in   preparing 
tracings  on  oiled  paper,  on  which  ink  collects  in  drops,  and  does  not  spread 
well.     If  the  paper  be  first  treated  with  a  little  ox-gall  and  allowed  to  dry  the 
difficulty  is  removed,  owing  to  the  reduction  in  surface  tension. 

4.  A  method  for  estimating   bile  salts  in    urine    has   been  described  by 
Griinbaum,  depending  on  this  property.     The  rate  of  escape  of  the  urine  from 
standard  capillary  tubes  is  noted,  the  rate  increasing  with  the  concentration  of 
bile  salts. 


118  THE    CONSTITUENTS     OF     BILE.  [CH.    VIII. 

225.  Oliver's  test  for  bile  salts.  Acidify  5  c.c.  of  the 
solution  with  two  or  three  drops  of  strong  acetic  acid,  filtering  if 
necessary.  To  the  acid  solution  add  an  equal  quantity  of  1  per  cent, 
solution  of  Witte's  peptone.  A  white  milkiness  or  a  decided 
precipitate  is  produced,  insoluble  in  excess  of  acid. 

NOTES. — 1.     The  precipitate   formed  consists  of   a  compound  of  protein 
with  bile  acids. 

2.     The  test  can  be  applied  to  urine.     (Ex.  288.) 

The  Bile  Pigments. 

Bilirubin,  C32H36N4O6,  is  a  reddish-brown  pigment 
most  abundant  in  the  bile  of  carnivora.  It  is  readily 
oxidised  by  the  oxygen  of  the  air  into  biliverdin,  C82H86N4O8, 
the  green  pigment  found  mostly  in  the  bile  of  "herbivora. 
These  compounds  are  formed  in  the  liver  cells  from  the 
products  of  disintegration  of  haemoglobin.  Haematiii  is 
C32H32N4O4Fe,  and  haematoporphyrin  is  isomeric  with 
bilirubin. 

They  are  weak  acids,  forming  sodium  and  calcium 
salts,  the  latter  being  insoluble  in  water.  Free  bilirubin 
is  soluble  in  ether  and  chloroform:  the  sodium  compound 
is  insoluble,  as  is  free  or  combined  biliverdin. 

By  oxidation  bilirubin  is  converted,  through  a  num- 
ber of  ill-defined  bodies,  such  as  bilicyaniri,  and  bilifusciii, 
into  choletelin,  the  end  product  of  Gmelin's  reaction. 

By  further  oxidation  a  compound,  haematinic  acid 
(C8H8O5),  is  formed,  identical  with  the  product  obtained  by 
the  oxidation  of  haematin  or  haematoporphyrin. 

By  reduction  with  sodium  amalgam  in  alcoholic 
solution  the  bile  pigments  are  converted  into  hydro- 
bilirubin,  which  is  also  formed  by  the  action  of  more 
powerful  reducing  reagents  on  haematin  or  haemato- 
porphyrin. 


CH.    VIII.]  THE     BILE     PIGMENTS.  119 

These  facts  all  indicate  the  close  relationship  between 
haematin  and  the  bile  pigments. 

In  the  bowel  the  bacteria  first  reduce  bilirubin  to 
hydrobilirubin.  This  is  then  attacked,  two  nitrogen 
atoms  being  probably  removed,  the  result  being  the 
formation  of  stercobilin,  which  is  mainly  excreted  in  the 
faeces.  But  a  small  amount  is  absorbed  and  excreted  in 
the  urine  as  urobilinogen. 

226.  Gmelin's   test   for   bile  pigments.     Take  a  few  c.c. 
of  fuming  yellow  nitric  acid  in  a  test  tube  and  by  means  of  a  pipette, 
carefully    place  on  the   surface   of  this   an  equal   amount  of   bile. 
Shake  the  tube  very  gently  from  side  to  side,  and  note  the  play  of 
colours  in  the  bile  as  it  becomes  oxidised  by  the  acid.     Proceeding 
from  acid  to  bile  the  colours  are  yellow,  red,  violet,  blue,  and  green. 

NOTES.— This  test  can  be  modified  in  many  ways. 

1.  Add  a  drop  of  yellow  nitric  acid   to  a  thin  film  of  bile  on  a  white 
porcelain  plate.     The  drop  of  acid  becomes  surrounded  by  rings  of  the  various 
colours. 

2.  Filter  some  diluted  bile  repeatedly  through  an  ordinary  filter  paper, 
and  then  place  a  drop  of  fuming  nitric  acid  on  the  paper.     The  play  of  colours 
is  usually  well  seen. 

227.  Cole's  test  for  bile  pigments.     To  about  50  c.c.  of 
diluted  bile  add  an  excess  of  baryta-mixture.'    Stir  well,  heat,  and 
allow  to  stand  for   a  short  time.     The  precipitate,  containing  an 
insoluble  barium  compound  of  bilirubin,  coheres  together.     Remove 
the  main  mass  of  the  fluid  by  means  of  a  pipette,  and  then  filter. 
Open  the  filter  paper  on  a  tile  and  scrape  the  precipitate  off  the 
paper.     Place  it  in  a  test-tube,  add  about  4  c.c.  of  strong  alcohol, 
two  drops  of  strong  sulphuric  acid,  two  drops  of  a  5  per  cent,  solution 
of  potassium  chlorate,  and  boil  for  a  minute.     Allow  the  precipitate 
of  barium  sulphate  to  settle.     The  supernatant  alcohol  is  coloured  a 
greenish-blue. 


120  THE     CONSTITUENTS    OF    BILE.  [CH.    VIII. 

NOTES. — 1.  If  the  precipitate  obtained  by  the  baryta-mixture  is  very  slight, 
a  small  amount  of  sodium  phosphate  solution  should  be  added  to  increase  the 
bulk  of  the  precipitate. 

2.  This   test   is   especially  valuable   for   detecting    the    presence   of   bile 
pigments  in  urine. 

3.  The  test  is  a  modification  of  one  originally  described  by  Huppert. 

The  Protein  of  Bile. 

When  bile  is  treated  with  acetic  acid  a  precipitate  is 
formed  insoluble  in  excess.  This  was  formerly  thought 
to  be  mucin.  But  it  has  been  shown  that  it  is  nucleo- 
protein,  the  bile  salts  present  preventing  the  re-solution 
in  strong  acetic  acid.  (See  Ex.  225.)  In  human  bile,  how- 
ever, mucin  is  present  as  well  as  nucleoprotein. 

The  protein  is  secreted  by  the  cells  lining^jbhe  ducts 
and  the  gall  bladder,  so  that  bile  from  the  gall  bladder 
contains  a  much  greater  percentage  than  fistula  bile. 

228.  To  a  small  quantity  add  strong  acetic  acid,  drop  by  drop. 
A  precipitate  is  formed,  insoluble  in  excess  of  acid.  This  precipi- 
tate consists  of  a  nucleoprotein,  together  with  a  considerable 
amount  of  the  bile  salts  and  bile  pigments. 

NOTE. — The  protein  precipitated  was  formerly  supposed  to  be  mucin, 
owing  to  the  insolubility  in  excess  of  acid.  It  has,  however,  been  shown  that 
it  is  a  nucleoprotein,  the  insolubility  in  excess  of  acid  depending  on  the  presence 
of  the  bile  salts.  (See  Ex.  288.)  It  is  generally  spoken  of  as  "  pseudo-mucin . " 

Human  bile  contains  both  mucin  and  nucleoprotein. 

Cholesterin.  C27H48OH  or  C27H45OH  is  a  monovalent 
alcohol  found  in  the  bile.  It  is  present  in  nearly  all  the 
fluids  and  tissues  of  the  body,  notably  in  the  central 
nervous  system.  It  is  found  in  large  amounts  in  egg- 
yolk.  In  the  blood  plasma  it  is  present  as  an  ester,  as  it 
is  in  lanoline,  the  "  fat "  obtained  from  sheep's  wool.  We 
have  already  seen  that  it  is  a  constituent  of  the  envelope 
of  red  blood  corpuscles  (p.  103).  It  forms  one  of  the 
varieties  of  gall  stones,  found  after  inflammation  of  the 
mucous  membrane  of  the  gall  bladder. 


CH.    VIII.]  CHOLESTERIN.  121 

It  is  soluble  in  ether,  alcohol,  chloroform,  and  acetone- 
It  is  only  slightly  soluble  in  cold,  easily  in  hot  alcohol. 
It  is  soluble  in  bile  salts,  insoluble  in  water,  weak  acids 
and  alkalies.  It  crystallises  from  boiling  alcohol  in  plates 
of  a  characteristic  shape :  from  the  other  solvents  in 
needles.  It  melts  at  145°C.,  and  in  chloroform  solution 
shows  an  optical  activity  [a]  D  =  -  36*6°. 

Its  chemical  constitution  is  not  yet  determined,  but 
it  probably  belongs  to  the  terpene  series. 


Preparation. — Sheep's  brain  is  minced,  ground  with  sand  and  inti- 
mately mixed  with  three  times  its  weight  of  plaster  of  Paris.  After  some 
hours  the  hard  mass  is  ground,  and  extracted  three  times  in  the  mortar 
with  acetone.  The  acetone  extract  is  filtered  and  evaporated  spon- 
taneously. The  cholesterin  crystallises  out,  and  can  be  recrystallised 
from  hot  alcohol. 


229.  Mount  a  few  crystals  of  cholesterin  in  water,  examine 
under  the  microscope,  and  draw  them.     Note  the  rhombic  plates,, 
which  are   often  incomplete  at  one  corner.     Irrigate  the  crystals 
with  strong  sulphuric  acid :  they  turn  red  at  the  edges.     Now  add 
a  drop  of  iodine  solution  :  the  crystals  give  a  violet  colour,  changing 
to  a  green,  blue,  and  finally  a  black. 

230.  Salkowski's  reaction  for  cholesterin.     Dissolve  a  little 
in  a  few  c.c.  of  chloroform  ;    to  the  solution  add  an  equal  quantity 
of  strong  sulphuric  acid  and  shake.     The  upper  layer  of  chloroform 
becomes    red,    the    layer    of   sulphuric    acid    yellow  with   a   green 
fluorescence. 

231.  Liebermann-Burchard     reaction     for     cholesterin. 

Dissolve  a  little  cholesterin  in  2  c.c.  of  chloroform,  contained  in  a 
perfectly  dry  tube.  Add  ten  drops  of  acetic  anhydride,  then  two 
drops  of  strong  sulphuric  acid,  and  shake.  The  solution  becomes- 
coloured  a  deep  blue. 


122  THE    CONSTITUENTS    OF    BILE.  [CH.    VIII. 

Lecithin  is  a  complicated  fat-like  body,  generally 
found  in  the  body  and  elsewhere  with  cholesterin.  (See 
p.  121.) 

It  can  be  regarded  as  a  compound  of  the  base  choline 
with  esters  of  glycerophosphoric  acid. 

CH2  -  OOC.C17H35  CH2  -  OOC.C17H;V) 

CH  -  OOC.C17H85  CH  -  OOC.Cl7Ht, 


CH0- 


CH2  -  O  CH2  -  O 

HO-P  =  O  HO  -  P  = 

HO  0    C2H4-0 

Di-stearyl  g lycerophosphoric 


acid.  o 

r3 

O 


2J 

^(OHJ, 


I 
OH 


Lecithin. 


CHAPTER    IX. 
URINE    AND    ITS    CHIEF    CONSTITUENTS. 

A.     The  average  composition. 

The  composition  of  the  urine  varies  with  the 
individual  and  with  the  diet.  Below  are  given  the 
figures  in  grams  for  the  daily  output  of 

A.  The  average  man  on  the  average  mixed  diet. 

B.  An  individual  on  a  liberal  diet. 

C.  The  same  individual  on  a  diet  deficient  in  proteins. 
B.  and  C.  are  taken  from  a  paper  by  Folin. 


A. 

B. 

C. 

d 

% 

«MOJ 

th 

02  ^\ 

*H   ^ 

d)  -g 

*£ 

Nitrogen. 

Per  cent,  of 
Total  N.  or  S. 

1 

| 

2 

i\ 

L 

^  a 

0)  4^ 

*8 

Urea 

30 

u 

87-5 

31-6 

14-7 

87-5 

4-72 

2-2 

61-7 

Ammonia 

0-6 

0-5 

3-1 

•6 

0-49 

3-0 

•51 

0-42 

11-3 

Creatinine 

1-55 

0-57 

3-6 

1-55 

0-58 

3-6 

1-61 

0-60 

17, 

Uric  Acid 

0-8 

0-23 

1-4 

•54 

0-18 

1-1 

•27 

0-09 

2-5 

Undetermined 

0-7 

4-4 

0-85 

4-8 

0-27 

7-3 

Total—  N 

16-0 

1000 

16-8 

100-0 

3-6 

100-0 

Inorganic  SO3 

2-92 

88-2 

3-27 

90-0 

0-46 

60-5 

Ethereal  SO» 

•22 

6-6 

0-19 

5-2 

0-10 

13-2 

Neutral  SO8 

•17 

5-2 

0-18 

4-8 

0-20 

26-3 

Total  SO3 

SI 

100-0 

3-64 

100-0 

0-76 

100-0 

124  URINE.  [CH.    IX. 

B.     The  Physical  Chemistry  of  the   Urine. 
1.     General  Properties. 

Normal  human  urine  is  a  clear  yellowish  fluid,  the 
depth  of  the  tint  depending  largely  on  the  concentration. 
On  standing,  a  cloud  (nubecula)  of  mucoid  containing 
epithelial  cells  separates  out.  After  a  heavy  meal  urine 
may  be  passed  cloudy,  due  to  earthy  phosphates  and 
carbonates.  On  standing,  these  settle  to  the  bottom  of 
the  vessel  as  a  white  deposit,  insoluble  on  warming,  but 
soluble  in  acids. 

Also  on  standing  a  cloud  of  urates  may  settle  as  a 
reddish  deposit  that  clears  up  on  warming. 

Fresh  urine  has  a  characteristic  odour  of  the  aromatic 
type,  due  to  the  presence  of  some  substance  that  has 
not  yet  been  recognised.  On  standing,  an  unpleasant 
ammoniacal  odour  develops  as  the  result  of  bacterial 
decomposition. 

II.     The   Specific   Gravity. 

Usually  lies  between  1012  and  1024  (water  =  1000). 
With  copious  drinking  it  may  fall  to  1002.  After  excessive 
perspiration  it  may  rise  to  1040. 

The  determination  of  the  specific  gravity  for  clinical 
purposes  is  most  conveniently  made  by  means  of  a  urino- 
meter,  a  weighted  cylinder  that  floats  in  the  urine.  The 
depth  to  which  it  sinks  depends  on  the  density  of  the 
fluid,  and  this  can  be  read  directly  by  means  of  a 
graduated  scale  on  the  stem.  The  instrument  is  calibrated 
for  a  certain  temperature,  usually  15°  C. 

The  urine  should  be  either  cooled  or  warmed  to  this 
temperature,  or  a  correction  made  by  adding  1  unit  for 
every  3  degrees  above  this,  or  subtracting  1  for  every  3 


CH.    IX.] 


THE     SPECIFIC     GRAVITY. 


125 


degrees  below  the  standard.     Thus  if  the  reading  be  1018 
at  18°  C.,  the  corrected  Sp.  Gr.  is  1019. 

To  obtain  the  best  results  two  separate  instruments 
should  be  at  hand,  the  one  calibrated  from  1000  to  1020 
and  the  other  from  1020  to  1040. 

The  total  amount  of  solids  in  the  urine  can  be  roughly  calculated 
from  the  specific  gravity  by  Long's  coefficient.  The  last  two  figures  of  the 
specific  gravity  x  2-6  gives  total  solids  in  1000  c.c. 

Thus  specific  gravity  at  25°  C.  =  1017. 

Total  solids  in  1000  c.c.  =  17  x  2-6  =  44-2  gms. 

Haser's  coefficient  (2-33)  on  a  similar  basis,  but  calculated  for  15°  C.  is 
probably  inaccurate. 


232.  Take  the  specific  gravity  of  normal  urine  by 
means  of  a  urinometer.  Wipe  the  instrument  clean, 
and  float  it  in  the  centre  of  a  cylinder  containing  the 
urine.  Remove  all  froth,  by  means  of  filter  paper  or  by 
placing  a  single  drop  of  ether  on  the  surface  of 
the  urine.  Take  care  that  the  instrument  does  not 
touch  the  sides  of  the  vessel.  Place  the  eye  level  with 
the  surface  of  the  fluid  and  read  the  division  of  the 
scale  to  which  the  latter  reaches.  Read  the  level  of  the 
true  surface  of  the  urine,  not  the  top  of  the  meniscus 
.around  the  stem. 


=  40- 


Fig.  3. 
Urinometer. 


126  URINE.  [CH.    IX. 

III.     The  Osmotic  Pressure  (Cryoscopijj. 

The  freezing  point  of  pure  water  is  0°C.  That  of 
solutions  is  lower  than  this,  and  the  depression  of  the 
freezing  point  is  proportional  to  the  molecular  concentra- 
tion of  the  solution.  In  the  case  of  electrolytes  (salts, 
alkalies  and  acids)  in  aqueous  solution  it  is  proportional 
to  the  concentration  of  (molecules  +  ions),  that  is  to  the 
"  osmotic  concentration." 

Since  the  osmotic  pressure  of  a  solution  is  also  pro- 
portional to  the  molecular  or  osmotic  concentration  of  the 
solution,  it  follows  that  a  determination  of  the  depression 
of  the  freezing  point  (cryoscopy)  enables  us  to  get  a 
measure  of  the  osmotic  pressure. 

With  non-electrolytes  the  gram-molecule  in  1000  gms. 
of  water  causes  a  depression  (A)  of  the  freezing  point  of 
1-85°  C. 

So  that  -^-Q=  =  molecular  concentration. 

I'OO 

With  electrolytes,  T-^-  =  osmotic  concentration  =  con- 

I'OO 

centration  (molecules  +  ions). 

The  quantitative  relationship  between  A  and  osmotic 
pressure  is  that  a  A  of  0-001°  C.  =  an  osmotic  pressure  of 
9-1  mm.  mercury. 

In  urine  the  concentrations  of  certain  substances> 
such  as  urea,  are  much  greater  than  they  are  in  the  blood. 
The  work  done  by  the  kidney  in  effecting  this  concentra- 
tion can  be  calculated  from  a  consideration  of  the  osmotic 
concentration,  i.e.  A,  of  each  substance  in  blood  and  urine. 
It  is  quite  erroneous  to  imagine  that  the  work  done  can 
be  calculated  from  a  knowledge  of  the  total  osmotic  con- 
centration of  the  blood  and  urine  respectively.*  But,  at 

*  A  full  discussion  of  the  subject  will  be  found  in  Moore's  article 
in  "Kecent  Advances  in  Physiology"  (p.  159). 


CH.    IX.]  THE     OSMOTIC     PRESSURE.  127 

the  same  time,  the  determination  of  A  of  the  blood  and  of 
the  urine  secreted  by  each  kidney  in  certain  renal  diseases 
may  give  us  valuable  information  as  to  the  relative 
activities  of  the  two  organs. 

A  of  blood  is  about  0-55°  C.,  the  same  as  that  of  a  0-9 
per  cent,  solution  of  sodium  chloride. 

A  of  urine  varies  considerably  with  the  diet,  volume  of 
fluid  taken  and  other  conditions.  For  the  mixed  24  hours 
urine  of  an  average  man  it  is  usually  about  1-2°  C.  The 
following  values  are  of  interest  in  this  connection  :— - 

.  A  x  volume  of  urine  =  molecular  diuresis. 

^-7^ T  is  of  considerable  pathological  signi- 

NaCl  per  cent. 

ficance.  It  is  fairly  constant  in  health,  varying  between 
1-25  and  1-6.  It  exceeds  1-7  in  heart  disease  or  in  any 
condition  that  causes  a  retardation  of  the  renal  circula- 
tion. The  only  febrile  condition  in  which  it  is  less  than 
1-7  is  malaria. 

233.  The  determination  of  the  freezing  point  by  Beck- 
mann's  method.  In  the  outer  chamber  (C)  place  a  mixture  of  ice 
and  water.  Add  saturated  salt  solution  until  the  temperature  falls 
to  about  3°  C.  lower  than  the  anticipated  freezing  point  of  the  urine. 
During  the  course  of  the  experiment  the  freezing  mixture  must  be 
stirred  occasionally  by  means  of  F,  and  ice  or  salt  added  to  main- 
tain its  temperature  within  about  1°C.  of  the  original. 

In  the  tube  A  place  enough  distilled  water  to  cover  the  bulb 
of  the  Beckmann  thermometer  D.  This  is  graduated  to  l/100th0  C., 
and  can  be  read  by  means  of  a  magnifying  glass  to  1/1000°  C. 
The  thermometer  must  not  touch  the  sides  or  bottom  of  the  tube  A, 
The  tube  B  serves  as  an  air  jacket  to  A.  Stir  the  water  regularly 
by  means  of  the  platinum  stirrer  E.  The  temperature  falls,  and 
then  after  a  time  rises  sharply,  and  remains  steady  for  a  con- 


128 


URINE. 


[CH.    IX. 


siderable  time.     The  temperature  to  be  read  is  the  highest  obtained 
at  this  rise.     This  is  the  freezing  point  (W)  of  distilled  water. 

Now  replace  the  water  by  the  urine,  rinsing  the  tube  out  with 
it  once  or  twice.  Repeat  the  experiment  and  note  the  freezing 
point  (F)  as  before.  W  --  F  =  A. 


Fig.  4.    Beckmann's  freez- 
ing point  apparatus. 


Fig.  5. 
Beckmann's 

Ther- 
mometer. 


NOTES. — 1.    It  is  of  the  utmost  importance  to  take  care  to  prevent  too  great 
a  super-cooling  of  the  urine.    This  should  never  exceed  1°C.    If  it  has  exceeded 


CH.    IX.  j  ACIDITY    OF    URINE.  129 

this  in  a  preliminary  experiment,  it  must  be  repeated,  and  when  the  temperature 
has  fallen  0'5eC.  below  the  freezing  point,  a  minute  crystal  of  ice  must  be 
introduced  through  the  side  tube.  These  crystals  are  best  prepared  by  taking 
in  a  dry  test-tube,  some  hollow  glass  beads  (that  have  been  carefully  dried) 
adding  a  small  amount  of  urine,  pouring  off  the  excess  of  fluid,  and  immersing  the 
tube  in  a  freezing  mixture.  They  should  be  introduced  by  means  of  a  pair  of 
cooled  forceps. 

2.  The  observed  A  is  usually  too  great,  owing  to  the  super-cooling.     The 

simplest  correction  is 

/          C\ 
A  corrected  =  A  observed  x    I  1  —  ^  I 

where  C  —  the  super-cooling  in  degrees. 

3.  To  set  the  thermometer.     Turn  the  thermometer  upside  down  and  by 
gentle  shaking  mix  the  mercury  in  the  upper  portion  with  that  in  the  capillary 
tube.    Then  place  the  thermometer  in  water  at  about  2°C.     Give  a  slight  knock 
and  thus  break  the  mercury  column.     It  is  now  ready  for  use. 

4.  When   reading    the  thermometer  during   an  experiment  it  should  be 
tapped  with  a  piece  of  indiarubber  tubing. 

IV.    Acidity. 

It  has  been  shewn  that  in  all  aqueous  solutions  the 
product  of  the  concentration  of  hydrogen  ions  (CH)  and 
that  of  the  hydroxyl  ions  (Cj^)  is  constant.  That  is 
CH  x  Cj~H  =  a  constant. 

In  distilled  water  at  18°  C.  these  concentrations  are 
equal  and  are  both  10  ~         So  that  the  constant  =  10  ~ 
x  10  ~    }l  =  10  ~        .      In    solutions   of   acids   CH  exceeds 
10  ~7'07  and  COH  is  less  than  10  "  '°',  but  the  product  of  the 

i  i  /\  -  14-14 

two  is  always  10 

Acids  differ  markedly  in  the  degree  to  which  they  are 
ionised  in  solution.  Thus  in  N/10  hydrochloric  acid  91  per 
cent,  of  it  is  ionised.  So  CH  is  0-091  N.  Now  0-091  =  9-1 


It  is  convenient  to  express  this  as  pn  =  1-04.     That  is, 
pH  is  the  logarithm  to  the  base  10  of  the  concentration  of 


130  URINE.  [CH.    IX. 

H  ions  in  grams  per  litre,  the  negative  sign  being  under- 
stood. 

N/10  acetic  acid  is  only  dissociated  to  the  extent  of  1-3 
per  cent. 

So  Cj  is  -0013  N  =  1-8  x  10  ~3  =  10  'n  "  *  =  10  ~2*a 

That  is  pH  =  2-89. 

An  "  indicator "  is  a  substance  that  shows  a  change 
in  colour  when  a  certain  amount  of  an  acid  or  an  alkali 
is  added  to  it.  At  a  certain  stage  of  the  addition  there 
is  an  intermediate  tint,  and  the  solution  is  now  said 
to  be  ''neutral  to  that  indicator."  It  must  be  clearly 
understood  that  this  so-called  neutrality  does  not 
necessarily  correspond  to  an  equality  in  the  concentration 
of  H  and  oli  ions.  Further,  a  solution  that  is  neutral 
to  one  indicator  may  have  a  concentration  of  H  ions 
widely  different  from  that  in  a  solution  that  is  neutral  to 
another  indicator.  Thus  a  solution  neutral  to  phenol- 
phthalein  has  a  pn  —  about  9 :  one  neutral  to  methyl- 
orange  has  pH  =  about  4.  The  value  pH  for  any  solution 
can  be  determined  electrically  by  means  of  the  potential  set 
up  between  the  solution  and  hydrogen.  Further,  it  has 
been  shown  that  if  two  solutions  show  the  same  tint  with 
a  given  indicator  at  about  the  neutral  point  of  this  in- 
dicator, then  these  solutions  have  the  same  pn-  Sorensen 
has  evolved  a  method  of  determining  the  true  acidity  of 
solutions  based  on  this  principle.  The  pH  is  roughly 
found  by  the  addition  of  various  indicators.  Then  a 
series  of  solutions  is  prepared  with  known  values  of  pn- 
A  certain  indicator  is  added  to  each  and  to  the  solution. 
Those  that  have  exactly  the  same  tint  have  equal  values 
of  PH- 

For  the  details  of  the  application  of  the  method  to 
urine,  the  student  should  consult  a  paper  by  G.  S. 
Walpole,  Bio-chemical  Journal,  Vol  V.,  p.  207. 


CH.    IX. j  ACIDITY    OF    URINE.  131 

The  range  of  certain  indicators  is  given  below. 

PH 
Methyl   violet  0-1  3-2 

Tropaeolin  OO  14  —      2-6 
Di-methyl-amino-azo-benzene 

(Topfer's  reagent)  2-9  4-2 

Methyl   Orange  3-1  44 

Methyl   Red  4-2  —      6-3 

p-Nitrophenol  5-0  7-0 

Litmus  5-0  8-0 

Neutral  Red  6-8  —      8-0 

Rosolic   Acid  6-9  8-0 

a-Naphtholphthalein  7-3  8-7 

Phenolphthalein  8-3  10-0 

Thymolphthalein  9-3  10-5 

Tropaeolin   O  11-1  12-7 

Normal  urine  has  pn  about  5,  that  is,  it  is  acid  to 
litmus  and  phenolphthaleiii,  but  alkaline  to  methyl  orange. 

The  amount  of  N/10  sodium  hydroxide  that  must  be 
added  to  make  the  mixture  neutral  to  pnenolphthalein 
is  sometimes  called  its  "  acidity."  It  would  be  better  to 
call  this  the  "titration  acidity."  For  the  method  of  its 
determination  see  Ex.  317. 

The  acidity  of  normal  urine  is  due  partly  to  the 
presence  of  acid  phosphates,  but  largely  to  free  organic 
acids. 

C.     The  Pigments  of  Urine. 

Urochrome  is  the  chief  pigment  of  normal  urine. 
It  is  a  yellow  substance  which  has  no  definite  absorption 
band.  Nothing  certain  is  known  as  to  its  constitution  or 
origin,  except  that  it  is  apparently  not  derived  from  the 
bile  pigments.  It  has  marked  reducing  properties. 


132  URINE.  [CH.    IX. 

Urobilin  occurs  in  fresh  normal  urine  as  its  chromogen, 
urobilinogen.  This  is  converted  into  urobilin  by  acids 
or  by  the  action  of  light  and  oxygen.  The  amount 
excreted  is  markedly  increased  in  fevers,  in  diseases  of 
the  liver  and  bile  passages,  by  destruction  of  the  red 
corpuscles,  especially  in  pernicious  anaemia,  and  during 
the  absorption  of  blood  clots.  In  certain  of  these  cases 
the  urobilin  itself  is  found  in  the  urine,  and  can  be 
identified  by  its  characteristic  absorption  band,  urobilin- 
ogen not  giving  a  definite  band. 

Urobilinogen  is  a  pyrrol  body  and  is  responsible  for 
Ehrlich's  reaction  with  p-dimethyl-amino-benzaldehyde. 

The  origin  of  urobilin  from  the  bile  pigments  is 
discussed  on  page  119.  It  may  be  added  that  tne  urobilin 
absorbed  from  the  bowel  into  the  circulation  is  mostly 
excreted  by  the  liver  into  the  bile,  so  that  only  a  small 
portion  reaches  the  urine.  Should  the  liver  cells  be 
injured  there  is  a  marked  increase  in  the  excretion  of 
either  urobilin  or  urobilinogen  in  the  urine. 

Uroerythrin  is  found  in  small  amounts  in  normal 
urine.  It  is  increased  in  fever  and  certain  diseases  of 
the  liver. 

It  is  soluble  in  amyl  alcohol.  Solutions  have  a 
reddish  colour,  but  are  unstable  to  light. 

The  pigment  is  usually  associated  with  the  urates 
or  uric  acid  of  the  urine. 

Haematoporphyrin  is  found  in  traces  in  normal  urine. 
There  is  a  certain  increase  in  fevers,  and  some  other 
diseases,  but  a  very  marked  increase  in  certain  cases  of 
poisoning  by  sulphonal  or  trional,  especially  in  women. 

Urorosein  occurs  in  urine  as  a  chromogen  which  is 
converted  into  the  pigment  by  the  action  of  strong  acids, 
such  as  HC1. 


CH.    IX. I  INORGANIC     CONSTITUENTS.  133 

It  is  insoluble  in  ether  and  is  thus  distinguished  from 
indigo  blue  formed  in  the  test  for  indican.  (Ex.  304.) 

The  chromogeii  seems  to  be  an  indol  body,  possibly 
indol-acetic  acid. 

234.  Note  the  colour  of  normal  urine  and  examine  some  in  a 
beaker    by    the    spectroscope.     Note    that    there    are    no    definite 
absorption  bands,  but  a  general  absorption  of  the1  violet.    Urochrome, 
the  chief  urinary  pigment,  yields  no  bands. 

235.  Saturate    at    least    200    c.c.    of    urine    with   ammonium 
sulphate.     Filter  off  the  precipitate  and  let  it  dry  completely  in  the 
air.     Extract  it  with  a  small  amount  of  strong  alcohol.     A  brownish 
solution  containing  urobilinogen  is  obtained.     Treat  this  with  a  few 
drops  of  hydrochloric  acid  :  the  urobilinogen  is  converted  to  urobilin. 
Examine  with  the  spectroscope,  and  note  a  single  absorption  band 
situated  at  the  junction  of  the  blue  and  the  green.     Its  centre  is 
about  A  490. 

D.     The  Inorganic  Constituents. 

Kations. 

Sodium  and  potassium  are  found  to  the  extent  of  3-2  gm. 
K2O  and  5-23  gm.  Na2O  per  diem.  The  ratio  K2O :  NayO 
generally  equals  1 : 1-54. 

During  starvation  this  can  rise  as  high  as  3 : 1,  owing 
to  the  excretion  of  the  potassium  of  the  tissues,  sodium 
being  found  in  a  much  smaller  amount  than  potassium. 
The  same  is  found  in  all  wasting  diseases. 

Calcium  and  magnesium  are  mainly  excreted  by  the 
bowel.  The  amounts  in  urine  are  0-33  to  0-6  gm.  CaO  and 
0-16  to  0-24  gm.  of  MgO. 

The  amounts  of  these  alkaline  earths  in  the  urine  are 
increased  by  the  administration  of  organic  acids,  or  in 
conditions  such  as  diabetes  in  which  the  formation  of  such 
acids  is  increased. 


134  URINE.  [CH.    IX. 

Iron  also  is  mainly  excreted  by  the  bowel.  It  is  found 
in  human  urine  only  in  organic  combination,  and  then 
only  to  the  extent  of  0-5  to  10  milligrams  per  diem. 

Anions. 

Chlorides  form  the  chief  part  of  the  anioiis  of  the  urine. 
The  amount  excreted  is  often  calculated  as  if  it  all  existed 
as  NaCl,  though  the  amount  of  sodium  in  the  urine  is 
normally  not  sufficient  to  combine  with  all  the  chlorine. 
The  amount  in  the  urine  depends  largely  on  the  amount  in 
the  food,  but  since  an  important  function  of  the  kidney  is 
to  maintain  a  constant  osmotic  pressure  of  the  tissue 
fluids,  mainly  by  variations  in  the  amount  of  NaCl 
excreted,  it  follows  that  anything  tending  to  cause  a 
change  in  the  osmotic  equilibrium  in  the  body  is  liable  to 
alter  the  excretion  of  chlorides  in  the  urine. 

Thus  during  starvation  and  during  the  formation  of 
exudates  in  pneumonia  the  chlorides  may  disappear 
from  urine.  The  amount  of  Cl  excreted  per  diem  is  about 
7  gms.  Reckoned  as  NaCl  it  is  12  grams. 

For  the  method  of  estimation  see  Ex.  318. 

Sulphates.  Only  a  small  portion  of  the  sulphate 
excreted  in  the  urine  is  taken  in  as  such  with  the  food. 
The  greater  portion  is  derived  from  the  oxidation  of  sulphur 
containing  substances,  chiefly  proteins.  The  amount 
of  sulphates  is  thus  a  rough  measure  of  the  total  amount 

N  5 

of  protein  metabolised,  the  ratio  -^~-  being  usually  — 

Sulphates  are  excreted  very  rapidly  after  a  protein 
meal,  reaching  a  maximum  about  the  third  hour.  This 
seems  to  indicate  that  cystine,  the  sulphur  complex  of 
proteins,  is  split  off  and  absorbed  very  early  in  the  diges- 
tion of  proteins. 


CH.    IX.]  ETHEREAL    SULPHATES.  135 

Ethereal  sulphates  are  esters  formed  by  the  union  of 
sulphuric  acid  with  phenols. 

O    OH  O    O.CaH, 

\/  "\/ 

S          +     HO.C6H6    —     =>        S  +     HaO 

v/\ 

O    OH  O    OH 

Sulphuric  acid.  Phenol.  Phenyl  sulphuric  acid. 

The  proportion  of  the  sulphur  that  is  present  as  ethereal 
sulphate  varies  considerably.  Folin  has  shewn  that  in 
starvation  and  on  diets  relatively  deficient  in  proteins  the 
proportion  increases,  as  does  that  of  the  "  neutral " 
sulphur.  There  is  also  a  marked  increase  after  the 
administration  of  certain  phenolic  substances,  or  when 
such  compounds  are  formed  in  the  body  by  bacterial 
decomposition,  as  in  intestinal  obstruction  and  severe 
constipation.  In  such  cases  the  phenols  found  conjugated 
with  sulphuric  acid  are 

C6H5.OH phenol    ) 

CH,(1)  T  r  formed  from  tyrosiiie. 

C6H4  C^OH  (4)   p-cresol  j 

C8H6N.OH iiidoxyl,  formed  from  tryptophane. 

These  bodies  are  poisonous.  They  unite  with  sulphuric 
acid,  probably  in  the  liver,  to  form  the  innocuous  ethereal 
sulphates. 

The  ethereal  sulphates  form  soluble  barium  salts,  and 
can  be  separated  from  the  inorganic  sulphates  by  treat- 
ment with  barium  chloride  and  filtering.  They  are 
hydrolysed  to  the  phenol  and  sulphuric  acid  by  boiling 
with  hydrochloric  acid. 

"  Neutral "  Sulphur.  In  urine  there  is  always  present 
a  certain  amount  of  sulphur  in  a  form  less  oxidised  than 
that  of  a  sulphate.  The  exact  nature  of  the  compounds 
in  urine  containing  sulphur  in  this  form  is  not  yet  clear. 


136  URINE.  [CH.    IX. 

It  is  probable  that  the  amount  of  "  neutral "  sulphur 
in  the  urine  is  independent  of  the  total  amount  of  sulphur 
excreted.  It  probably  varies  with  the  amount  of  tissue 
protein  metabolised,  so  that  its  determination  is  often  of 
considerable  interest. 

For  the  percentages  of  sulphur  excreted  in  the  three 
forms  under  different  metabolic  conditions  see  page  123. 

For  the  methods  of  determination  of  the  sulphur  see 
Exs.  320-322. 

Phosphates.  The  phosphates  of  the  urine  are  present 
on  the  one  hand  as  salts  of  the  alkali  metals  and  of 
ammonium  ;  on  the  other,  as  salts  of  the  alkaline  earths, 
calcium  and  magnesium.  About  3-9  grms,  of  P2O5  are 
excreted  per  diem  in  the  urine.  Phosphoric  acid  forms 
three  series  of  salts.  The  formulae  for  that  of  sodium  and 
calcium  are 

Normal  phosphate,  Na3PO4  :  Ca8(PO4)2. 

Mono-hydrogen  phosphate,  Na.2HPO4  :  CaH(PO4). 
Di-hydrogen  phosphate,  NaH2PO4        :  CaH4(PO4)2. 

The  three  sodium  salts  and  CaH,(PO4)2  are  soluble  in 
water:  the  other  two  calcium,  salts  are  insoluble.  The 
normal  and  mono-hydrogen  phosphates  are  alkaline  in 
reaction  to  litmus  :  the  di-hydrogeii  phosphates  are  acid. 

The  phosphates  of  the  urine  are  derived  partly  from 
the  inorganic  phosphates  of  the  food,  partly  from  the 
oxidation  of  phosphorus-containing  substances  of  the 
food  and  tissues,  such  as  nucleo-proteins,  lecithins  and 
phospho-proteins,  and  partly  also  from  the  phosphates  of 
bone.  The  exact  share  played  by  these  various  compounds 
in  forming  the  urinary  phosphates  is  difficult  to  deter- 
mine owing  to  the  fact  that  a  proportion  of  the  phosphates, 
varying  between  12  and  50  per  cent.,  are  excreted  by  the 


CH.    IX.]  PHOSPHATES.  137 

bowel.  In  this  connection  it  may  be  noted  that  alkaline 
phosphates  of  the  food  are  more  likely  to  be  excreted  in 
the  urine  than  are  earthy  phosphates. 

The  excretion  of  varying  amounts  of  phosphates  by 
the  kidney  is  one  of  the  methods  by  means  of  which  the 
reaction  of  the  body  fluids  is  maintained  in  equilibrium. 
An  increased  excretion  is  always  seen  in  cases  of  acid 
poisoning  and  in  the  acidosis  associated  with  diabetes. 

As  soon  as  the  urine  shews  a  certain  grade  of 
alkalinity,  a  precipitation  of  earthy  phosphates  takes 
place.  This  is  sometimes  known  as  phosphaturia,  but  it  is 
not  necessarily  associated  with  an  increase  of  phosphates 
in  the  urine.  In  the  phosphaturia  of  juveniles  it  is 
probable  that  there  is  an  excessive  amount  of  calcium  in 
the  urine,  due  to  a  defective  excretion  of  the  large 
intestine. 

A  certain  amount  of  phosphorus  is  found  in  the 
urine  in.  an  organic  form,  not  as  a  phosphate.  It  may  be 
present  as  glycero-phosphoric  acid.  The  average  daily 
amount  is  about  50  mgms. 

For  method  of  estimation  see  Ex.  319. 

236.  Test  for  chlorides  by  adding  to  about  3  c.c.  of  urine  a 
few  drops  of  pure  nitric  acid  and  3  c.c.  of  a  3  per  cent,  solution  of 
silver  nitrate.     An   abundant   curdy  precipitate   of   silver  chloride 
appears  at  once.     If  the  chlorides  are  less  in  quantity,  the  solution 
merely  becomes  milky  or  opalescent. 

NOTE.— If  nitric  acid  is  not  added,  urates  might  be  precipitated  by  silver 
nitrate,  especially  if  the  urine  be  ammoniacal. 

237.  To  a  test  tube  nearly  full   of   urine  add  a  little  strong 
ammonia  and  boil.     A  white  flaky  precipitate  of  the  phosphates 
of  calcium  and  magnesium  is  formed.     Filter  off  the  precipitate, 
wash  with  water,  and  dissolve  in  5  c.c.  of  dilute  acetic  acid.     Divide 
the  solution  into  two  parts.    To  one  part  add  a  solution  of  potassium 


138  URINE.  [CH.    IX. 

oxalate.     A  white  precipitate  is  produced,  showing  the  presence  of 
calcium  in  the  urine. 

238.  To  the  other  portion  of  the  solution  add  an  equal  bulk 
of  strong  nitric  acid  and  about   5   c.c.  of    ammonium    molybdate. 
Boil :    a  yellow   crystalline    precipitate    is    produced,   showing  the 
presence  of  phosphates. 

NOTE. — Neutral  urine  is  very  apt  to  yield  a  precipitate  of  earthy  phosphates 
on  boiling,  owing  to  the  change  of  reaction  due  to  the  evolution  of  CO-2  (See 
notes  to  Ex.  9). 

239.  To   demonstrate   the   presence  of  acid-phosphates 
in  urine.    Treat  5  c.c.  of  urine  with  ah  equal  volume  of  5  per  cent, 
solution   of    barium   chloride.      Filter   repeatedly   through  a  small 
filter   paper   till  the    nitrate    is    clear.     Treat    the   filtrate    with    a 
little  baryta  mixture  and  boil.     Filter  ;  dissolve  the  precipitate  in 
nitric  acid  and  boil  the  solution  obtained  with  ammonium  molybdate. 
The  yellow  precipitate  shows  the   presence   in    the    urine    of  acid 
phosphates,  such  as  NaH2PO4. 

NOTE. — Any  alkaline  phosphate,  Na2HPO4,  present  in  the  urine  is  precipi- 
tated by  BaCl2  as  BaHPO4.  The  acid  phosphates  remain  in  solution  as 
Ba(H2PO4)2.  On  the  addition  of  the  alkaline  baryta  mixture,  the  acid  phosphate 
is  converted  into  the  insoluble  alkaline  phosphates  of  barium.  If  no  precipitate 
is  produced  when  the  baryta-mixture  is  added,  there  are  no  acid  phosphates 
present  in  the  sample  of  urine. 

Since  the  acidity  of  a  sample  of  urine  varies  almost  directly  with  the  amount 
of  acid  phosphates  present,  as  determined  by  the  above  method,  it  is  generally 
held  that  the  acidity  of  urine  is  mainly  due  to  the  presence  of  these  acid 
phosphates. 

240.  Treat  10  c.c.  of  urine  with  a  few  drops  of  strong  hydro- 
chloric acid,  and  about  3  c.c.  of  a  solution  of  barium  chloride.     A 
precipitate  of  barium  sulphate  is  produced  as  an  opaque  milkiness. 
If  the  precipitate  is  thick  the  sulphates  are  in  excess.     (The  hydro- 
chloric acid  is  added  to  prevent  the  precipitation  of  phosphates.) 

241.  To  demonstrate  the  presence  of  ethereal  sulphates. 

To  urine  add  an  equal  bulk  of  baryta  mixture  (two  parts  of  baryta 
water  to  one  part  of  a  10  per  cent,  solution  of  barium  nitrate).  A 
precipitate  is  formed  consisting  of  the  phosphates  and  the  ordinary 
inorganic  sulphates.  Filter  till  quite  clear.  To  the  filtrate  add  a 


CH.    IX.]  UREA.  139 

third  of  its  volume  of  strong  hydrochloric  acid,  boil  in  a  beaker  for 
five  minutes,  and  allow  to  stand.  A  faint  white  cloud  of  barium 
sulphate  is  formed  indicating  the  presence  of  ethereal  sulphates  in 
the  urine. 

NOTES.  —  1.  The  ethereal  sulphates  form  soluble  barium  salts,  but  are 
hydrolysed  to  sulphuric  acid  by  heating  with  an  acid. 

C6H5  -  O^ 

^>S02  +  H20=C6H5.OH  +  H2S04. 
HO  Phenol. 

Phenyl-sulphuric  acid. 

The  sulphuric  acid  thus  formed  is  converted  into  barium  sulphate  by  the 
excess  of  barium  present. 

2.  The  solution  becomes  very  dark  in  colour  on  boiling  with  the  strong 
acid,  owing  to  the  action  of  the  latter  on  the  aromatic  chromogenic  substances 
in  the  urine. 

E.    Urea. 

Urea  is  the  compound  in  which  the  greater  part  of 
the  nitrogen  is  normally  excreted  in  man.  The  percent- 
age of  the  urinary  nitrogen  in  the  form  of  urea  varies. 
Normally  it  is  about  90  per  cent.,  but  in  starvation,  or  on 
a  diet  deficient  in  proteins,  it  is  only  about  60  per  cent.  It 
is  also  low  in  cases  of  diabetes  accompanied  by  acidosis 
(owing  to  the  relatively  high  percentage  of  ammonia),  and 
also  in  certain  cases  of  hepatic  disorder,  notably  acute 
yellow  atrophy  of  the  liver,  owing  to  the  non-formation 
of  urea  by  the  disordered  liver,  its  seat  of  formation  in 
the  body. 

The  total  amount  excreted  per  diem  by  a  normal  man 
on  an  average  diet  containing  100  grams,  of  protein  is 
30  grams. 

Urea  is  also  known  as  carbamide,  since  it  is  the  di- 
amide  of  carbonic  acid. 


0    r_  _  OH 

-OH  -NH2 

Carbonic  acid.  Urea. 

Urea  crystallises  in  water-free,  colourless,  long 
needles,  or  in  four-sided  prisms  of  the  rhombic  system, 
which  melt  and  decompose  at  130  -  132°  C. 


140  URINE.  [CH.    IX. 

It  is  soluble  in  all  proportions  in  hot  water,  and  to 
the  extent  1 : 1  in  cold  water.  In  cold  alcohol  it  is  soluble 
to  the  extent  of  1:5.  It  is  also  soluble  in  acetone. 
Insoluble  in  pure  ether  and  chloroform.  The  solutions 
are  neutral  in  reaction. 

It  forms  crystalline  compounds  with  acids.  The  two 
most  important  are  urea  nitrate  CH4  N2O.  HNO3,  insoluble 
in  strong  nitric  acid,  and  urea  oxalate  (CH4  N2O)2,  C2H2O4, 
insoluble  in  oxalic  acid. 

It  forms  compounds  with  the  salts  of  the  heavy 
metals,  especially  with  mercuric  nitrate  (see  below,  Ex.  250). 

With  reducing  sugars  relatively  stable  compounds 
are  formed,  called  ureides.  They  are  of  importance  in 
connection  with  the  estimation  of  urea  in  diabetic  urine. 

On  heating  dry  urea  to  140°  C.,  ammonia  is  evolved  and 
biuret  formed. 

NH2 

NH, 
CO 

CO 
NH, 

NH        +     NHS 

io 

CO 

NH2 
NH.2 

Biuret. 

On  heating  beyond  140°  C.,  cyanuric  acid  and  ammonia 
are  formed.  Cyanuric  acid  is  C3H3N8O3. 

N 


HO— C    C— OH 


N 
C— OH 


CII.    IX.]  UREA.  141 

Solutions  of  urea  are  decomposed  by  boiling  alkalies 
into  CO2  and  NH8.  They  are  also  similarly  decomposed 
by  heating  for  several  hours  at  150°  C.  with  acids.  This 
decomposition  is  readily  effected  by  the  addition  of 
magnesium  chloride,  zinc  sulphate  or  potassium  acetate 
to  the  solution  for  the  purpose  of  raising  the  boiling 
point. 

Bacteria,  as  micrococcus  ureae,  decompose  urea  into 
CO2  and  NH3.  This  accounts  for  normal  urine  rapidly 
becoming  ammoniacal  on  standing  in  the  air. 

Nitrous  acid  decomposes  urea  as  follows : — 
CO(NH2)2  +  2HN02  =  2N2  +  CO2  +  3H2O. 

Hypobromites  effect  a  similar. decomposition. 

CO(NH2)2  +  SNaBrO  =  3NaBr  +  CO2  +  N2  +  2H2O 

Sodium 
hypobromite. 

242.  To  a  watch-glass  half  full  of  distilled  water  add  as  much 
solid  urea  as  will  lie  on  a  sixpenny-piece.     Note  the  solubility  of 
urea  in  water. 

243.  Place  a  drop  of  the  urea  solution  on  a  slide,  add  a  single 
drop  of  a  saturated  solution  of  oxalic  acid,  mix  by  stirring  with  a 
needle  or  fine  glass  rod,  cover  with  a  slip  and  examine  the  crystals 
of  oxalate  of  urea  that   separate   out.     They   vary   considerably, 
containing    long,  thin,  flat  crystals,  often  in  bundles  and  rhombic 
prisms.     Draw  the  crystals. 

244.  Dilute  the  urea  solution  with  twice  its  volume  of  water. 
Place  a  drop  on  a  slide,  add  a  drop  of  pure  nitric  acid,  cover  with  a 
slip,  and  examine  the  crystals  of  urea  nitrate  that  separate  out. 
They  form  octahedral,  lozenge-shaped,  or  hexagonal  plates,  often 
striated  and  imbricated.     Draw  the  crystals. 

245.  Powder  two  or  three  crystals  of  urea  in  a  watch-glass  : 
rub  with  a  small  amount  of  acetone  and  warm  gently  on  a  water 
bath.     The  urea  dissolves.     Allow  most  of  the  acetone  to  evaporate 
away,  and  then  place  a  drop  of  the  remaining  solution  on  a  watch- 


142  URINE.  [CH.    IX. 

glass.     Urea  crystallises  out  as  the  acetone  passes  off.     Draw  the 
crystals. 

246.  Repeat  the*  above  exercise,  using  strong  alcohol  instead 
of    acetone.     Draw    the    crystals  of  urea,  which  are  usually  very 
irregular. 

247.  Dilute  the  remainder  of  the  aqueous  solution  left  from 
Ex.  244  with  an  equal  quantity  of  water,  and  to  a  portion  of  this  in 
a  test  tube  add  some  yellow  nitric  acid  (or  nitric  acid  to  which  a 
little    potassium    nitrite    has    been    added).     An  effervescence  and 
evolution  of  gas  take  place. 

CO(NH2)2  +  2HNO2  =  CO2  +  2N2  +  3H2O. 

NOTE. — All   compounds   containing    the   amido   group   (NH2)    react    in  a 
similar  manner  when  treated  with  nitrous  acid. 

248.  To    another    portion  of  the  solution  add  sodium  hypo- 
bromite.     A  marked  effervescence  and  evolution  of  gas  take  place. 

CO(NH2)2  +  SNaBrO  +  2NaHO 

=  3NaBr  +  Na2CO3  +  3H2O  +  N2. 

249.  To    a   few    c.c.    of    saturated  ammonium  sulphate   add 
sodium  hypobromite.     A  marked  effervescence  and  evolution  of  gas 
take  place. 

(NH4)2SO4  +  SNaBrO  +  2NaHO 

=  Na2SO4  +  5H2O  +  3NaBr  +  N2. 

NOTES — 1.     All  ammonium  salts  and  all  compounds  with  the  amido  group 
give  off  nitrogen  when  treated  with  an  alkaline  solution  of  sodium  hypobromite. 

2.  The  sodium  hypobromite  is  prepared  as  follows  :  dissolve  100  grams, 
of  caustic  soda  in  250  c.c.  of  water.     Cool,  and  slowly  add  25  c.c.  of  bromine, 
cooling  under  the  tap  as  the  bromine  is  added.     The  reaction  is  as  follows  : 

2  NaHO  +  Br2  =  NaBrO  +  NaBr  +  H.^O. 

It    must    be  freshly  prepared  before  use   as   it    undergoes   the   following 
decomposition 

3  NaBrO  -  2  NaBr  +  NaBrO3. 

3.  As  a  test  for  urea  the  reaction  with  hyprobromite  is  only  useful  in  a 
negative  sense  ;  that  is  to  say,  if  an  effervescence  is  not  obtained  urea  is  absent, 
but  if  an  effervescence  is  obtained  it  does  not  necessarily  follow  that  urea  is 
present. 

250.  To  some  of  the  urea  solution  add  a  solution  of  mercuric 
nitrate.     A  white  precipitate  of  mercuric  oxide  combined  with  urea 


CH.    IX.]  UREA.  143 

and  mercuric  nitrate  takes  place.  To  the  mixture  thus  obtained 
add  a  saturated  solution  of  sodium  chloride,  drop  by  drop.  The 
precipitate  dissolves,  to  reappear  on  a  further  addition  of  mercuric 
nitrate. 

NOTES. — 1.  The  precipitate  consists  of  urea  and  mercuric  nitrate  and  one, 
two  or  three  molecules  of  mercuric  oxide,  depending  on  the  concentration  of 
the  two  solutions. 

2.  The  solubility  in  NaCl  is  due  to  the  formation  of  mercuric  chloride,, 
which  is  only  very  feebly  ionised  in  neutral  solutions. 

3.  The  reaction  is  sometimes  useful  in   detecting  the  presence  of  urea  in 
solutions.     Proteins  give  a  precipitate  with  mercuric  salts,  which  is  soluble  or 
insoluble  in  NaCl,  depending  on  the  nature  of  the  protein.     Therefore,  to  make 
the  test  more   certain,   proteins  should  be  removed  by  the  method  given  in 
Ex.  55.     Since  phosphates  give  a  very  similar  reaction  they  must  be  removed 
by  baryta  mixture  before  testing  for  urea  (See  Ex.  255). 

251.  Treat  a  solution  of  urea  with  Millon's  reagent,  and  heat. 
A  white  precipitate  is  formed,  owing  to  the  presence  of  mercuric 
nitrate  in  the  reagent.     There  is  also  an  evolution  of  gas  due  to  the 
action  of  the  nitrous  acid  on  the  urea. 

252.  Boil  1  c.c.  of  a  dilute  solution  of  urea  with  a  little  strong 
alkali  for  fifteen  minutes.     Cool,  neutralise  with  diluted  sulphuric 
acid  and  test   for  urea  by  the  addition   of  mercuric  nitrate.     No. 
precipitate  is  obtained  owing  to  the  hydrolysis  of  the  urea  by  the 
boiling  alkali. 

CO(NH2),  +  H2O  =  COa  +  2NH». 

253.  Place  a  little  urea  in  a  dry  test  tube.     Heat  carefully 
over  a  flame,  keeping  the  upper  part  of  the  tube  cool.     The  urea 
melts  and  evolves  ammonia,  whilst  a  white  sublimate  condenses  on 
the  cooler  parts  of  the  tube.     Cool  the  tube,  add  a  little  water  and 
shake.     Pour  the  solution  into  another  tube  and  treat  it  with  an 
equal  bulk  of  sodium  hydroxide  and  a  drop  of  copper  sulphate.     A 
pink  colour  is  produced,  due  to  the  biuret  formed  from  the  urea. 

254.  Repeat  the  experiment,  but  heat  more  strongly  till  the 
melt  solidifies  and  becomes  opaque.     Cool,  add  two  or  three  c.c.. 
of  water,  boil  and  filter  whilst  still  hot.     Divide  the  solution  into 
two  portions  A  and  B.     To  A  add  a  few  drops  of  a  solution  of 


144  URINE.  [CH.    IX. 

barium  chloride  and  a  single  drop  of  diluted  ammonia.  A  white 
mass  of  barium  cyanurate  is  formed  on  cooling. 

To  B  add  some  ammoniacal  copper  sulphate  solution  and  boil. 
On  cooling  an  amethyst  precipitate  of  copper  ammonium  cyanurate 
is  deposited. 

NOTE. — Preparation  of  ammoniacal  copper  sulphate.  1  per  cent,  copper 
sulphate  is  treated  with  very  dilute  ammonia  till  the  precipitate  that  first  forms 
just  redissolves. 

255.  To   demonstrate   the   presence   of   urea    in    urine. 

Treat  5  c.c.  of  urine  with  half  its  bulk  of  baryta  mixture,  and  filter 
•off  the  precipitate  of  sulphates  and  phosphates.  Neutralise  the 
filtrate  with  acetic  acid  and  add  a  little  mercuric  nitrate.  A  white 
precipitate,  soluble  in  sodium  chloride,  is  obtained,  indicating  the 
presence  of  urea.  (See  Ex.  250.) 

NOTE.— Phosphates  must  be  removed,  as  they  give  a  similar  reaction. 

256.  Isolation  of  urea  from  urine.     Evaporate  about  30  c.c. 
of  urine  to  complete  dryness,  finishing  the  evaporation  on  the  water 
bath  (to  prevent  the  destruction  of  the  urea).     Turn  out  the  flame 
and  rub  the  residue  with  about   10  c.c.  of  acetone  till  it  is  boiling. 
Allow  the  acetone  to  boil,  stirring  all  the  time,  till  about  half  of  it 
has  evaporated  away.     Pour  off  the  acetone  into  a  dry  watch  glass 
and  allow  it  to  cool.     Crystals  of  urea  separate  out  as  silky  needles. 
Demonstrate  that  they  are  urea  crystals  by  evaporating  to  dryness, 
taking  up  in  a  small  amount  of  water  and  obtaining  characteristic 
•crystals  of  urea  nitrate.     (See  Ex.  244). 

F.     Uric  Acid. 
Uric  Acid,  C5H4N4O3,  is  2r6-8-tri-oxy-purine. 

NH  -  CO 

I 


O       C  -  NH 

II 
H  -  C  - 


Its   relationship  to  certain  of  the   other   purines   is 
indicated  on  page  20. 


CH.    IX.]  URIC    ACID.  145 

When  pure  it  crystallises  in  microscopic  rhombic 
plates,  hut  when  impure  it  assumes  a  variety  of  forms, 
such  as  whetstones,  dumb-bells,  sheaves,  rosettes, 
butchers'  trays,  etc. 

It  dissolves  to  the  extent  of  1  part  in  16,000  parts  of 
cold  water  and  1600  parts  of  hot  water.  It  dissolves  in 
alkalies,  and  the  alkali  salts  of  carbonic,  phosphoric,  boric, 
lactic  and  acetic  acids,  but  not  in  the  ammonium  salts  of 
these  acids.  It  dissolves  in  warm  concentrated  sulphuric 
acid  to  form  a  sulphate,  which  is  decomposed  by  the 
addition  of  water. 

It  is  precipitated  by  phosphotungstic  acid  in  the 
presence  of  hydrochloric  acid,  slowly  by  lead  acetate,  and 
completely  by  picric  acid,  mercuric  chloride  and 
ammoniacal  silver  nitrate. 

By  oxidation,  allantoin,  alloxan,  parabanic  acid  and 
urea  are  formed,  depending  on  the  reaction  and  the 
reagent  employed. 

NH2  NH  -  CO  NH  -  CO 

CO      CO  -  NIL,  CO      CO  CO 

NH  -  CH  -  NH^  NH  -  CO  NH  -  CO 

Allantoin.  Alloxan.  Parabanic  acid. 

Although  the  aqueous  solutions  of  uric  acid  react 
neutral,  it  behaves  like  a  disbasic  acid  C5H2N4O8.H2  and 
can  form  two  series  of  salts,  C5H2N4O3.Na2  (neutral, 
normal,  or  di-sodium  urate)  and  C5H2N4O3.HNa  (biurate, 
acid  urate  or  mono-sodium  urate).  It  is  also  possible  that 
there  is  a  third  form  of  salt,  C5H2N4O3.HNa.C5H,N4O3 
(quadriurate  or  hemi-sodium  urate),  though  this  may  be 
merely  a  mixture  of  its  two  constituents.  The  di-sodium 
salts  are  more  soluble  than  the  mono-sodium,  but  are 


146  URINE.  [CH.    IX. 

only  stable  in  markedly  alkaline  solutions.  In  the  blood 
and  urine  urates  exist  as  mono-sodium  salts,  which 
react  neutral. 

It  is  interesting  to  note  that  there  are  two  modifi- 
cations of  the  mono-sodium  salt,  called  the  a-  and  /3-form. 
The  a-form  is  more  soluble  than  the  /3-form,  but  is  un- 
stable, and  slowly  passes  over  into  the  other  form.  They 
are  probably  the  salts  of  the  two  tautomeric  modifications 
of  uric  acid  described  by  Fischer : 

NH  -  CO  N  -  C.OH 


CO       0  -  NH^  HO.C 

Jr 


C-NH, 

H.CO >  >CO 

-  C  -  NIK  N  -  C  -  NEK 

Lactam  modification  forming  Lactim  modification  forming 

unstable  a-urate.  stable  /3-urate. 

It  is  of  great  interest  to  observe  that  in  gout  the 
amount  of  urate  in  solution  in  the  blood  is  in  excess  of 
the  amount  of  the  /3-urate  that  can  be  held  by  normal 
blood.  So  that  in  gout  it  must  be  present  at  least,  partly, 
in  the  unstable  a-form.  The  deposition  of  urates  in  the 
tissues  during  an  acute  attack  may  be  due  to  the  con- 
version of  the  unstable  a-  into  the  stable,  less  soluble 
^-modification. 

Urates  are  completely  precipitated  as  amorphous 
ammonium  urate  by  saturation  with  ammonium  chloride. 

They  exert  a  reducing  reaction  on  Fehling's  solution 
and  towards  alkaline  silver  solutions,  this  being  the  basis 
of  Schiff's  test. 

They  yield  a  characteristic  colour  reaction  when 
evaporated  with  nitric  acid,  the  so-called  murexide  test. 

Uric  acid  occurs  to  the  extent  of  about  0-7  gm.  in  the 
24  hours'  urine,  but  the  amount  excreted  varies  with  the 
diet  and  the  individual. 


CH.    IX  ORIGIN     OF     URIC    ACID.  147 

From  its  close  chemical  relationship  to  the  purine 
bases  formed  by  the  hydrolysis  of  the  nucleins  of  the  food 
and  tissues  (see  p.  20),  the  view  is  commonly  held  that 
uric  acid  has  its  origin  in  the  cellular  organs  of  the  body 
from  the  oxidation  of  such  substances.  Thus  we  can  have 
uric  acid  arising  exogenously  from  the  free  or  combined 
puriiies  of  the  food  and  also  eiidogenously  from  those  of 
the  tissues.  This  view  is  apparently  supported  by  the 
fact  that  the  administration  of  foods  rich  in  nucleoproteins, 
as  sweetbreads,  or  of  certain  of  the  pure  purine  bases, 
does  cause  an  increased  excretion  of  uric  acid. 

Plimmer  has  remarked  on  the  close  relationship 
between  the  elimination  of  uric  acid  and  the  number  of 
leucocytes  in  the  blood,  and  makes  the  suggestion  that 
uric  acid  is  a  product  of  the  metabolism  of  the  leucocytes. 
This  is  not  a  revival  of  the  old  theory  that  it  is  formed  by 
the  disintegration  of  these  cells. 

It  is  important  to  note  that  a  certain  proportion  of 
the  uric  acid  formed  in  the  body  is  destroyed  by  the  liver, 
so  that  the  amount  excreted  is  a  balance  between  that 
formed  and  that  destroyed. 

In  gout,  in  which  there  is  a  deposition  of  uric  acid  in 
the  tissues,  the  excretion  is  decreased  before  an  acute 
attack,  is  increased  during  the  attack,  and  then  falls  again. 
In  this  condition  there  is  a  recognisable  amount  of  uric 
acid  in  the  blood  (see  above).  For  methods  of  estimation 
in  urine  see  Exs.  314,  315. 

257.  Treat    a    small    amount    of    uric    acid    with    10  c.c.     of 
2   per  cent,   sodium  carbonate.     Heat  nearly  to  boiling  and  cool. 
Note  that  a  considerable  portion  of  the  uric  acid  has  dissolved  in 
the  form  of  a  urate. 

258.  Filter  the  solution  and  treat  a  portion  with  a  drop  or  two 
of  strong  hydrochloric  acid  and   shake.     A  white  crystalline  pre- 
cipitate of  uric  acid  separates  out,  showing  that  uric  acid  is  very 


148 


URINE.  [CH.    IX. 


insoluble  in  water.  Allow  the  crystals  to  settle,  remove  a  few  by 
means  of  a  pipette,  and  examine  them  microscopically.  They 
usually  form  rhombic  plates.  Draw  the  crystals. 

NOTE. — If  the  solution  is  very  strong,  the  uric  acid  may  separate  out  in  an 
amorphous  form.  Should  this  be  the  case,  make  the  solution  alkaline  and  heat 
to  dissolve.  Whilst  still  hot  add  some  HC1  and  allow  the  tube  to  cool  slowly. 

Uric  acid  can  assume  a  great  variety  of  crystalline  forms,  resembling  dumb- 
bells, whetstones,  butcher-trays,  stars,  and  sheaves. 

259.  To  another  portion  of  the   solution  add  two  drops  of 
ammonia  and  saturate  with  ammonium  chloride.     A  white  amor- 
phous precipitate  of  ammonium  urate  is  formed. 

NOTE. This  is  the  basis  of  Hopkins'  original  method  for  the  estimation  of 

urates  in  urine.  It  is  an  important  reaction  for  separating  urates  from  physio- 
logical fluids,  such  as  urine  (see  Ex.  268),  since  no  other  organic  substance,  likely 
to  be  met  with  in  physiological  analysis,  is  precipitated  by  saturation  with 
ammonium  chloride.  The  murexide  reaction  can  be  applied  to  the  precipitate 
obtained. 

260.  Treat  a  little  uric  acid  with  a  little  strong  sulphuric  acid  : 
it   dissolves.      Pour   the   solution   into   water :    the   uric   acid   may 
separate  out. 

261.  Murexide  test.      Treat  a  little  uric  acid  in  a  porcelain 
dish  with  two  or  three  drops  of  strong  nitric  acid.     Heat  on  the 
water-bath   till    every    trace    of    nitric    acid   and   water   has    been 
removed.     A  reddish  deposit  remains.     Treat   this   with   a    dilute 
solution  of  ammonia  (five  drops  of  ammonia  to  about  a  test  tube 
full  of  water).     The  residue  turns  reddish  violet  in  colour.     Add 
a  little  caustic  soda.     The  colour  turns  to  a  blue-violet. 

NOTES  1.— This  important  test  needs  a  certain  amount  of  care.  The 
heating  must  be  performed  on  the  water-bath  and  should  be  continued  as 
long  as  is  necessary  to  ensure  the  complete  removal  of  every  trace  of  nitric  acid. 

2  Xanthine  and  guanine  give  a  yellow  substance  (nitro-xanthine)  when 
treated  with  nitric  acid.     On  evaporation  the  colour  goes  to  a  violet  shade,  which 
turns    yellow   with    dilute   ammonia.      Adenine    and    hypoxanthine    give    no 
colour  reaction. 

3  The  chemistry   of  the  reaction  is  as  follows  :    From  uric  acid  arises 
by  oxidation  dialuric  acid  and   alloxan.        They  condense   together    to    form 


CH.    IX.]  TESTS    FOR    URIC    ACID.  149 

alloxantin.        By    the    action    of    ammonia  on   alloxantin,    purpuric    acid    is 
formed.     Murexide  is  ammonium  purpurate. 

HN-CO  HN-CO  HN-CO  OC-NH 

|        |       H  ||  |       |        OH  |        | 

OC     C^  +    OC     CO  OC     C/i  _  C      CO 


I       I    -  OH  ||  ||        HO^ 

HN-CO  HN-CO  HN-CO  OC-NH 

Dialuric  acid  Alloxan.  Alloxantin. 

HN-CO         NH       OC-NH 

I       I 
Alloxantin  +  NH3  =  OC      C: 

I        I 

HN-CO  OC-NH 

Purpuric  acid. 

262.  Schiff  S  test.     Treat  a  very  small  amount  of  uric  acid 
with  a  few  c.c.  of  sodium  carbonate.     Pour  the  solution  on  to  a 
filter  paper  moistened  with  silver  nitrate.     A  black  stain  of  reduced 
silver  immediately  results. 

NOTE.—  This  useful  test  cannot  be  applied  in  the  presence  of  chlorides.  It 
is  important  to  note  that  the  uric  acid  is  dissolved  in  sodium  carbonate,  not  the 
hydroxide,  as  the  latter  gives  a  precipitate  of  the  brown  silver  hydroxide,  which 
completely  obscures  the  reduction.  An  amount  of  sodium  carbonate  in  excess 
of  that  required  to  dissolve  the  uric  acid  must  be  added,  as  the  reduction  only 
takes  place  in  the  alkaline  condition. 

263.  Folin's  test.     To  a  very  small  pinch  of  uric  acid  in  a 
beaker  add  20  c.c.   of  a  saturated  solution  of  sodium  carbonate. 
Stir  till  the  uric  acid  has  completely  dissolved,  add  1  c.c.  of  Folin's 
uric  acid  reagent.     A  blue  colour  is  obtained. 

NOTES.  —  Preparation  of  Folin's  solution.  100  grams,  of  pure  sodium 
tungstate,  102  c.c.  of  pure  ortho-phosphoric  acid  (B.P.  66'3%)  and  750  c.c.  of 
distilled  water  in  a  flask  fitted  with  a  reflux  condenser  are  boiled  for  2  hours. 
On  cooling  the  solution  is  diluted  to  1  litre. 

2.  The  solution  also  gives  a  blue  colour  with  polyphenols.  It  is  used  for 
the  microchermcal  estimation  of  uric  acid  in  urine  (Ex.  315). 

264.  Dissolve  a  little  uric  acid  in  sodium  carbonate  by  boiling. 
Add  5  c.c.  of  Fehling's  solution  and  boil  for  a  considerable  time. 
Note  the  peculiar  reduction  of  the  copper,  and  compare  it  with  the 
reduction  obtained  with  glucose. 

265.  Similarly  try  the  effect  of  uric  acid  on  Nylander's  (Ex. 
70)  and  Benedict's  (Ex.  68)  solutions.     A  reduction  is  not  obtained. 


150  URINE.  [CH.    IX. 

266.  Dissolve    some    uric  acid  in  sodium  carbonate,  add  an 
excess  of  ammonia  and  treat  with  silver  nitrate.     A  white  amorphous 
precipitate  of  a  silver  compound  of  uric  acid  is  formed. 

NOTE. — Xanthine,  hypoxanthineand  other  substances  in  urine  closely  related 
to  uric  acid  are  similarly  precipitated  by  ammoniacal  silver  nitrate. 

267.  A  solution  of  sodium  urate  and  urea  is  provided. 
To  prepare  crystals  of  uric  acid  and  of  urea. 

Heat  a  test  tube  nearly  full  of  the  solution  to  boiling  point  and 
add  strong  hydrochloric  acid  till  the  reaction  is  distinctly  acid. 
Allow  the  tube  to  cool  slowly ;  the  uric  acid  crystals  separate  out. 
Cool  thoroughly  under  the  tap.  Filter  off  the  uric  acid.  Neutralise 
the  filtrate  with  sodium  carbonate  and  evaporate  to  dryness,  finishing 
the  process  on  the  water-bath,  to  prevent  the  conversion  of  the  urea 
to  biuret.  (See  Ex.  253.)  Extract  the  residue  with  strong  alcohol  or 
acetone.  The  alcohol  or  acetone  solution  is  carefully  evaporated  to 
dryness,  and  the  urea  crystallises  out. 

268.  To  demonstrate  the  presence  of  uric  acid  in  urine. 

Treat  50  c.c.  of  urine  with  two  drops  of  ammonia  and  then  stir 
with  powdered  ammonium  chloride  till  the  solution  is  saturated. 
Allow  the  excess  of  ammonium  chloride  to  settle  for  15  sees.,  and 
pour  off  into  another  beaker.  Note  the  gelatinous  precipitate  of 
ammonium  urate.  Filter  :  scrape  the  precipitate  off  the  paper  and 
transfer  it  to  an  evaporating  dish.  Add  three  or  four  drops  of  strong 
nitric  acid  and  place  the  dish  on  the  water  bath  till  a  pink,  dry 
residue  is  obtained.  Treat  this  with  a  little  dilute  ammonia :  the 
purple  colour  produced  indicates  the  presence  of  urates  in  urine. 
(See  Exs.  259  and  261.) 

269.  Folin's   method  of  demonstrating  the   presence  of 
uric  acid  in  urine.     To   1  to  2  c.c.   (20  drops)   of  urine  in  an 
evaporating  dish  add  one  drop  of  a  saturated  solution  of  oxalic  acid 
and  evaporate  to  complete  dryness  on  a  water  bath.     Allow  to  cool, 
add  10  c.c.  of  strong  alcohol  and  allow  to  stand  for  five  minutes  to 
extract  the  polyphenols.     Carefully  pour  off  the  alcohol.     To  the 
residue  add  10  c.c.  of  water  and  a  drop  or  two  of  saturated  sodium 


CH.    IX.]  PURINE    BASES.  151 

carbonate.  Stir  to  secure  complete  solution  of  the  uric  acid  and 
transfer  to  a  beaker.  Add  1  c.c.  of  Folin's  uric  acid  reagent  (Ex. 
263)  and  20  c.c.  of  saturated  sodium  carbonate  solution.  The  blue 
colour  that  results  indicates  the  presence  of  uric  acid. 

270.  Urine  has  been  treated  with  about  one-fiftieth  its  bulk 
of  strong  hydrochloric  acid,  and  allowed  to  stand  from  twelve  to 
twenty-four  hours.  Note  the  brown  crystals  of  uric  acid  that  have 
formed  on  the  sides  of  the  vessel.  Examine  them  microscopically  : 
they  form  very  irregular  crystals,  usually  arranged  in  sheaves. 
Draw  the  crystals. 

NOTE. — The  chief  pigment  that  associates  itself  with  uric  acid  and  urates  is 
known  as  uroerythrin.      (See  p.   132.) 

G.     Purine  bases,  other  than  uric  acid. 

The  most  important  of  these  found  in  normal  urine 
are  hypoxanthine,  xanthine  and  adenine  (see  p.  20), 
derived  from  the  metabolism  of  food  and  tissue  nucleins  : 
heteroxanthine  (7-methyl-xanthine)  and  paraxanthine 
(1,  7-dimethyl-xanthine)  derived  from  the  breakdown  of 
caffeine  (1,  3,  7-trimethyl-xanthine)  and  theobromine 
(3,  7-dimethyl-xanthine)  of  the  coffee,  tea  and  cocoa 
ingested. 

In  man  the  methylated  xanthines  constitute  the 
greater  part  of  these  purine  bases.  But  it  is  interesting 
to  note  that  the  non-methylated  ones  are  much  increased 
in  fever.  Also  during  severe  muscular  exercise  there  is  an 
increase,  accompanied  by  a  decrease  of  uric  acid.  After 
the  exercise  there  is  an  increase  of  uric  acid,  and  a 
decrease  of  the  other  purines. 

The  simplest  method  of  estimation  is  to  determine 
uric  acid  nitrogen  by  the  method  in  Exs.  314,  315,  and 
the  total  purine  nitrogen  by  applying  Kjeldahl's  method 
(Ex.  306)  to  the  total  purines  precipitated  by  ammoniacal 
silver  nitrate  (Ex.  266).  The  difference  is  the  nitrogen 
of  the  purine  bases. 


152  URINE.  [CH.    IX. 

H.    Greatinine  and  Greatine. 

The  chemical  relationships  of  these  bodies  are 
described  on  p.  77.  In  normal  human  urine  creatinine 
is  always  present,  but  creatine  only  after  a  meat  diet, 
being  derived  from  that  of  the  food.  Creatine,  however, 
is  a  normal  constituent  of  the  urine  of  children. 

Creatinine  seems  to  be  a  product  of  tissue  metabolism, 
and  the  amount  excreted  is  regarded  by  Polin  as  a 
measure  of  endogenous  metabolism.  (See  tables  B  andC, 
p.  123.)  There  is  an  increase  in  complete  starvation  and 
in  fevers,  due  to  the  increased  breakdown  of  the  tissues. 
Mellanby  has  drawn  attention  to  the  fact  that  the  liver 
is  probably  the  seat  of  formation  of  creatinine.  Thus 
in  most  diseases  of  the  liver  there  is  a  decreased  excretion, 
an  important  exception  being  hepatic  carcinoma,  in 
which  condition  the  urinary-creatinine  is  increased  and 
is  accompanied  by  creatine.  Creatine  is  excreted  when 
the  muscles  of  the  body  are  broken  down.  This  explains 
the  presence  of  creatine  in  urine  during  starvation  and 
in  fevers. 

When  creatinine  is  given  by  the  mouth  it  is  mainly 
excreted  unchanged,  but  a  small  portion  is  broken  down 
into  unknown  products.  When  creatine  is  administered 
it  also  is  chiefly  excreted  unchanged,  but  a  certain 
percentage  is  destroyed  in  the  body.  The  amount  excreted 
unchanged  is  considerably  increased  with  diets  rich  in 
proteins. 

Properties.  Creatinine  dissolves  in  11  parts  of  water 
and  102  parts  of  alcohol  at  16°  C.  It  is  insoluble  in  ether. 
Its  solutions  are  neutral  or  very  slightly  alkaline  in 
reaction. 

Creatinine  is  precipitated  by  phosphotuiigstic  acid, 
by  picric  acid,  and  by  the  salts  of  the  heavy  metals. 


CH.    IX.]  CREATININE.  153 

Alkalies  convert  it  slowly  into  creatine.  On  boiling 
with  barium  hydroxide  it  is  converted  into  urea  and 
sarcosine  (see  p.  77). 

Creatinine  reduces  Fehling's  solution,  but  not 
Benedict's  or  Nylander's  solutions. 

For  the  method  of  estimation  see  Ex.  316. 

271.  Jaffe's  test.      To  5  c.c.  of  urine  add  a  few  drops  of  a 
saturated  aqueous   solution  of  picric  acid  and  of  a    10   per  cent, 
solution    of    sodium    hydroxide.     A    red    colouration    is    produced 
owing  to  the  formation  of  picramic  acid. 

272.  Weyl's  test.     To  5  c.c.  of  urine  add  a  few  drops  of  a 
freshly    prepared    5    per   cent,    solution    of    sodium    nitroprusside. 
Add  a  5  per  cent,  solution  of  sodium  hydroxide,  drop  by  drop.     A 
ruby-red  colour  appears,  which  quickly  turns  yellow. 

NOTE. — Acetone  gives  a  similar  red  colour,  but  it  does  not  turn  yellow. 

273.  Salkowski's  test.     To  the  yellow  solution  obtained  in 
the  preceding  exercise  add  an  excess  of  acetic  acid  and  boil.     A 
greenish  blue  colour  results.     On  standing,  a  sediment  of  Prussian 
blue  may  separate. 

I.    Ammonia. 

Ammonia  is  a  constituent  of  normal  urine,  being 
present  to  the  extent  of  about  O7  gm.  per  diem.  There  is 
an  increased  excretion  following  the  administration  of 
ammonium  salts  of  inorganic  acids,  in  certain  cases  of 
hepatic  disease,  and  as  a  result  of  acid  poisoning.  This 
last  condition  ("  acidosis ")  can  be  produced  by  the 
administration  of  inorganic  acids  or  by  the  excessive 
formation  of  acids  in  the  body,  especially  if  this  is  not 
accompanied  by  an  increased  intake  of  alkalies.  Thus  it  is 
seen  in  severe  diabetes,  in  starvation,  and  in  delayed 
chloroform  poisoning,  the  acids  formed  being  aceto-acetic 
and  /3-oxy-butyric  acids. 

For  methods  of  estimation  see  Exs.  308  to  310. 


154  URINE.  [CH.    IX. 

J.    Hippuric  Acid. 

Hippuric  acid  is  formed  in  the  kidney  by  the  con- 
densation of  benzoic  acid  with  glycine. 

C6H5.COOH   +   H2N.CH2.COOH  C6H5.CO.NH.CH2COOH    +    H-/> 

Benzoic  acid.  Glycine.  Hippuric  acid. 

The  amount  excreted  by  a  normal  individual  on  a 
mixed  diet  is  about  -7  gm.  per  diem.  It  is  increased  by  a 
vegetable  diet,  owing  to  the  presence  in  most  plant  foods  of 
an  aromatic  complex  that  is  oxidised  to  benzoic  acid  in 
the  body. 

Hippuric  acid  crystallises  in  4-sided  prisms,  somewhat 
resembling  triple  phosphate.  It  melts  at  187-5°  C. :  above 
this  temperature  the  melt  becomes  red  and  Is  decom- 
posed into  benzoic  acid,  benzonitrile  and  prussic  acid.  It 
is  soluble  in  hot  water,  alcohol  and  ethyl  acetate : 
insoluble  in  benzene  and  petroleum  ether :  only  slightly 
soluble  in  cold  water,  alcohol,  ether  and  chloroform.  It 
forms  an  insoluble  ferric  salt.  By  hot  acids  or  alkalies 
it  is  hydrolysed  to  benzoic  acid  and  glycine.  When 
evaporated  with  strong  nitric  acid,  nitrobenzene  is 
formed. 

274.  Isolation  from  urine  by  Roaf  s  method.     500  c.c.  of 
the  urine  of  a  horse  or  cow  are  treated  with  125  grams  of  ammonium 
sulphate  and  7'5  c.c.  of  concentrated  sulphuric  acid.     On  standing 
for  24  hours  the  hippuric  acid  crystallises  out.     Filter  off  the  crystals, 
and  wash  with  a  little  cold  water.     Dissolve  in  a  small  amount  of  hot 
water,   boil    with   a   little    animal    charcoal,    filter,    concentrate    if 
necessary,  and  allow  to  stand  for  24  hours. 

275.  To  a  little  hippuric  acid  in  a  small  evaporating  dish  add 
1  to  2  c.c.  of  concentrated  nitric  acid  and  evaporate  to  dryness  in 
a  water-bath  in  the  fume  chamber.     Transfer  the  residue  to  a  dry 
test  tube,  apply  heat,  and  note  the  odour  of  nitrobenzene  (artificial 
oil  of  bitter  almonds). 


CH.    IX.]  ALBUMIN.  155 

276.  Neutralise  a  solution  of  hippuric  acid  with  dilute  caustic 
soda.     Add   a  few    drops    of    ferric    chloride.      A    cream-coloured 
precipitate  of  the  ferric  salt  of  hippuric  acid  is  formed. 

K.    Certain  Constituents  of  Abnormal  Urine. 

1.    Albumin  and  Globulin. 

"Albuminuria  "  is  the  name  given  to  the  condition  in 
which  a  heat-coagulable  protein  is  found  in  the  urine,  no 
matter  whether  the  protein  present  is  albumin  or  globulin. 
As  a  rule  both  proteins  are  present,  but  albumin  is  gener- 
ally greatly  in  excess  of  the  globulin. 

Albuminuria  can  be  renal  ("true")  or  accidental 
("false").  Renal  albumin uria  can  be  brought  about  by  an 
alteration  in  the  blood  pressure  in  the  kidney,  by  a  change 
in  the  composition  of  the  blood,  or  by  an  alteration  in  the 
structure  of  the  kidney.  In  accidental  albuminuria,  the 
protein  is  not  passed  by  the  kidney,  but  gains  access  to  it 
lower  down  in  the  urinary  tract.  It  is  generally 
accompanied  by  haemoglobiiiuria. 

For  the  method  of  estimating  the  albumin  see  Exs. 
323,  324. 

277.  Boiling  test.      Filter  the  urine  till  it  is  clear.     If  it  will 
not  filter  clear,  as  when  infected  with  bacteria,  shake  with  kieselguhr 
and  filter  again.      If  the  urine  be  alkaline  to  litmus,  make  it  faintly 
acid  by  the  cautious  addition   of  1   per  cent,  acetic  acid.     Fill  a 
narrow  test  tube  three  parts  full  with  the  clear  urine,  incline  it  at  an 
angle  and  boil  the  upper  layer  by  means  of  a  very  small  flame.     A 
turbidity  indicates  either  albumin  or  earthy  phosphates  (see  note 
2  to  Ex.  9).     Add  one  or  two  drops  of  strong  acetic  acic,  boiling 
after  the  addition  of  each  drop.     Any  remaining  turbidity  indicates 
the  presence  of  albumin. 

278.  Heller's  test.     Place  about  3  c.c.  of  pure  nitric  acid  in  a 
narrow  test  tube.     Float  about  3  c.c.  of  filtered  urine  on  the  surface 
of  this,  using  a  pipette  to    avoid    mixing.     A  white  ring  at  the 
junction  of  the  fluids  indicates  the  presence  of  albumin. 


156  URINE.  [CH,   IX. 

NOTES. — 1.  The  white  ring  is  due  to  the  formation  of  metaprotein  by  the 
action  of  the  acid  on  the  albumin,  and  the  insolubility  of  the  metaprotein  in  the 
strong  nitric  acid.  (See  Exs.  1,  13  and  42). 

2.  A  coloured  ring  is  usually  produced  owing  to  the  oxidation  of  certain 
urinary  chromogens. 

3.  In  very  concentrated  urine,  a  white  ring  of  urea  nitrate  may  form.     It 
usually  has  very  sharply  denned  borders. 

4.  If  the  urine  is  very  rich  in  urates,  a  precipitate  of  uric  acid  may  form 
at  the  junction  of  the  fluids,  or,  more  commonly,  somewhat  above  the  nitric 
acid.     Urea   and   uric   acid   are   distinguished  from  albumin   by    the  previous 
dilution  of  the  urine  with  two    or  three  volumes  of  water. 

5.  The  presence  of  resinous  substances  in  the  urine  of  patients  who  have 
been  treated  with  balsams  leads  to  the  development  of  a  white  ring  or  cloud 
that  disappears  on  treatment  with  alcohol. 

6.  Urine   rich  in  albumose  may  give  a  white  cloud  that  disappears  on 
warming. 

7.  Urine  that  has  been  preserved  by  the  addition  of  thymol  gives  a  ring  of 
nitrosothymol  or  nitrothymol.    The  thymol  can  be  removed  by  gentle  agitation 
with  petroleum  ether. 

279.  Roberts'    test.      Repeat    the    previous   exercise,  using 
Roberts'  reagent  in  place  of  the  nitric  acid.     A  white  ring  at  the 
junction  of  the  fluids  indicates  albumin. 

NOTES. — 1.  Roberts'  reagent  is  prepared  by  adding  1  volume  of  pure  nitric 
acid  to  5  volumes  of  a  saturated  solution  of  magnesium  sulphate. 

2.     Coloured  rings  are  not  formed,  and  so  confusion  is  avoided. 

280.  Spiegler's    test.       Render  the  urine   faintly   acid  with 
acetic  acid  and  repeat  the  above  test,  using  Spiegler's  reagent  in 
place  of  Roberts'.     A  white  ring  indicates  the  presence  of  albumin. 

NOTES  1.     Spiegler's  reagent  consists  of 

Mercuric  chloride         ...          ...          ...         40  gm. 

Tartaric  acid      ..          ...          ...          ...          20  gm. 

Glycerine  ...          ...          ...          ...        100  gm. 

Sodium  chloride  ...          ...          ...          50  gm. 

Distilled  water  1000  c.c. 

2.  The  reaction  is  also  given  by  albumoses  and  peptones. 

3.  The  test  serves  to  show  1  part  of  albumin  in  250,000.     It  is  almost  too 
delicate  for  ordinary  clinical  work,  as  a  large  number  of  apparently  normal 
urines  give  a  positive  reaction. 

2.    Albumoses. 

Albumoses  are  found  in  the  urine  in  certain  cases  of 
degeneration  of  the  intestinal  epithelium  ("alimentary 
albumosuria  ").  Also  in  a  variety  of  other  conditions  such 


CH.    IX.]  BENCE-JONES'     PROTEIN.  157 

as  in  the  absorption  of  pneumonic  exudates,  in  some  cases 
of  an  increased  breakdown  of  the  tissues  in  certain  fevers, 
in  the  puerperium,  and  in  urine  containing  semen. 

The  albumose  present  seems  to  be  a  secondary 
albumose. 

281.  Remove    any    albumin    that    may   be    present    by   heat 
coagulation.      To  the  filtrate  apply   Spiegler's  test  (Ex.  280).     A 
white  ring  indicates  the  presence  of  albomose. 

3.    Bence-Jones'   Protein. 

In  certain  cases  of  disease  of  the  bone  marrow  (multiple 
myeloma),  and  possibly  in  osteomalacia,  a  protein  with 
peculiar  properties  is  found  in  the  urine.  It  is  named 
after  Bence-Jones,  who  first  described  the  condition.  It 
has  the  property  of  coagulating  at  temperatures  under 
55°  C.,  of  redissolving  to  a  clear  solution  on  boiling  and 
of  reappearing  on  cooling.  It  is  precipitated  by  half- 
saturation  with  ammonium  sulphate.  It  is  not  precipitated 
on  dialysis. 

Hopkins  has  shewn  that  the  solution  of  the  heat 
coagulum  on  boiling  depends  on  the  presence  of  neutral 
salts,  those  with  divalent  cations  (as  CaCl2)  being  most 
potent  in  neutral  or  faintly  acid  solutions,  and  those  with 
divalent  anions  (as  K2S04)  in  faintly  alkaline  solutions. 

Hopkins  has  also  shewn  that,  the  protein  excreted  is 
formed  in  the  body,  either  in  the  marrow  or  as  a  result  of 
the  influence  of  the  growth  on  general  metabolism.  The 
amount  in  the  urine  is  independent  of  the  nature  or 
amount  of  the  proteins  of  the  food.  The  nitrogen  of  the 
protein  excreted  may  be  as  high  as  one-third  of  the  total 
urinary  nitrogen. 

282.  If  necessary  make  the  suspected  urine  faintly  acid  with 
acetic  acid.     Heat  carefully  by  immersing  in  a  beaker  of  warm 


158  URINE.  LCH.  ix. 

water.  The  urine  becomes  turbid  at  40°  to  45° C.,  and  shows  a 
flocculent  precipitate  at  60° C.  On  raising  the  temperature  to  100°C., 
the  precipitate  partially  or  completely  disappears.  On  cooling  it 
reappears. 

4.    Blood  Pigments. 

Blood  pigments  may  occur  in  pathological  urine  in 
intact  corpuscles  ("  haematuria ")  or  free  in  solution 
("  haemoglobinuria  "). 

Haematuria  can  be  recognised  by  determining  the 
presence  of  red  corpuscles  by  a  microscopic  examination 
of  the  sediment  obtained  by  centrifugalising  the  urine. 
It  occurs  with  gross  lesions  of  the  kidney  or  any  part 
of  the  urinary  tract,  so  that  blood  passes  directly  into 
the  urine.  If  the  blood  comes  from  the  kidney  it  is  well 
mixed  with  the  urine.  If  the  blood  comes  from  the 
bladder  or  genital  organs  it  often  forms  a  clot.  In 
haematuria  the  urine  often  has  a  characteristic  smoky 
appearance,  and  it  is  always  associated  with  albuminuria. 
Haemoglobinuria  is  a  result  of  haemolysis.  It  therefore 
follows  a  variety  of  infectious  diseases,  transfusion  of 
blood,  the  absorption  of  haemolytic  substances,  such  as 
many  aromatic  compounds,  severe  burns  and  scalds. 
Methaemoglobin  is  nearly  always  present. 

283.  Heller's    test.     Boil   10  c.c.   of   urine   with  a  little  40 
per  cent,  sodium  hydroxide,  and  allow  the  tube  to  stand  for  a  while. 
A  red  deposit  indicates  the  presence  of  blood-pigment  in  the  urine. 
Pour  off  the  supernatant  fluid  and  acidify  with  acetic  acid.     The 
precipitate  dissolves  only  partially,  leaving  a  red  residue. 

NOTES  1. — The  alkali  converts  the  pigment  into  haematin,  which  is  pre- 
cipitated with  the  earthy  phosphates. 

2. — Certain  substances,  such  as  cascara  sagrada,  rhubarb,  senna  and 
santonin  cause  the  urine  to  give  a  similar  red  precipitate  when  boiled  with 
alkali.  But  in  these  cases  the  precipitate  dissolves  completely  in  acetic  acid. 

284.  Schumm's  spectroscopic  test.     Treat   50  c.c.   of   the 
urine  with  5  c.c.  of  glacial  acetic  acid  and  50  c.c.  of  ether.     Shake 


CH.    IX.]  BILE.  159 

thoroughly  in  a  separating  funnel.  Allow  to  stand  and  add  a  drop 
or  two  of  alcohol  to  obtain  a  separation  of  the  layers.  Run  off  the 
urinary  layer.  To  the  ether  add  5  c.c.  of  water,  shake  and  run  off 
the  water.  To  the  washed  ether  add  ammonia  and  shake  for  half 
a  minute,  cooling  under  the  tap.  The  reaction  must  be  markedly 
alkaline  after  shaking.  Run  off  the  lower  coloured  layer  into 
a  tube,  add  5  to  10  drops  of  ammonium  sulphide  solution  and 
examine  spectroscopically  for  the  bands  of  haemochromogen. 
(Ex.  219.) 

285.  Adler's   benzidine   test.     To   a   saturated   solution  of 
benzidine    in   alcohol  or  glacial   acetic  acid  add  an  equal  bulk  of 
3   per    cent,    hydrogen    peroxide   find    1  c.c.  of  the   urine.     If  the 
mixture  is  not  acid,   render  it  so  by  the  addition  of  acetic  acid. 
The  appearance  of  a  green  or  blue  colour  indicates  the  presence  of 
blood  pigment. 

NOTES. — 1.    A  control   test   should    be  performed,  substituting  water  for 
the  urine. 

2.  The  reaction  can  be  applied  to  the  acid  ethereal  solution  prepared  in 
the  preceding  exercise. 

3.  Benzidine    preparations    vary     considerably     in     sensitiveness.      The 
solutions  must  be  kept  in  the  dark. 

5.    Bile. 

The  constituents  of  the  bile  are  found  in  urine  when 
the  bile  duct  is  obstructed  by  a  calculus  or  by  catarrh. 
The  bile  is  absorbed  into  the  lymphatics,  passes  into  the 
circulation  and  reaches  all  parts  of  the  body,  the  pigments 
causing  a  staining  of  the  various  tissues.  The  condition 
is  known  as-jaundice. 

The  absence  of  bile  salts  from  the  urine  does  not 
exclude  the  possibility  of  the  presence  of  bile  pigments. 
With  continued  obstruction  of  the  bile  passages  the 
formation  of  bile  salts  seems  to  decrease.  Urine  con- 
taining bile  often  has  a  characteristic  appearance. 

286.  Cole's  test  for  bile  pigments.     To  25  c.c.  of  urine  add 
baryta  mixture  and  proceed  as  directed  in  Ex.  227. 


160  URINE.  [CH.    IX. 

287.  Hay's   test   for   bile    salts.     Sprinkle   the   surface  of 
some  urine  in  a  test  tube  with  flowers  of  sulphur.     The  particles 
fall  to  the  bottom  of  the  tube  if  bile  salts  are  present.   (See  Ex.  224.) 

288.  Oliver's  test   for   bile   salts.     Acidify   the   urine   with 
acetic  acid  and  niter  if  necessary.     To  it  add  a  clear  1  per  cent, 
solution  of  Witte's  peptone,  also  acidified  with  acetic  acid.     A  white 
precipitate  indicates  bile  salts.     (Ex.  225.) 

289.  Jolle's  test  for  bile  salts.     Treat  50  c.c.  of  urine  with 
15  c.c.    of    a    3'  per    cent,    solution    of    casein,    add    10    per    cent, 
sulphuric    acid,    drop    by    drop,    with    continued   stirring  until  the 
casein    is  completely   precipitated   (-6   to   '8  c.c.   usually   required). 
Filter,    and    treat    the    precipitate  in  a   small   beaker  with    10  c.c. 
of  strong  alcohol.     Allow  to  stand  for  1  hour  at  room" -temperature, 
stirring   frequently.     Filter   and   treat    5  c.c.    of    the    filtrate    with 
one    drop    of    a    5    per   cent,   solution   of   rhamnose  and   5  c.c.   of 
concentrated    hydrochloric    acid.      Boil    over   a   small    flame,    and 
keep   gently   boiling   for   about   two   minutes.      Cool,    add    2  c.c. 
of  ether  and  shake.     A  characteristic  green  fluorescence  indicates 
the  presence  of  bile  salts. 

6.     Glucose. 

Glucose  seems  to  be  a  constituent  of  normal  urine, 
but  the  amount  present  is  very  small  (0-01  to  0-04  per  cent.). 
When  present  in  recognisable  quantities  the  condition  is 
known  as  glycosuria. 

There  are  two  types  of  glycosuria,  alimentary  and 
persistent.  Alimentary  glycosuria  is  the  condition  in 
which  the  amount  of  sugar  absorbed  exceeds  the  amount 
that  the  individual  is  capable  of  assimilating.  The  limit 
varies  with  the  individual,  and  is  affected  by  a  variety  of 
pathological  conditions.  Persistent  glycosuria  is  the 
condition  when  large  amounts  of  sugar  are  excreted  for  a 
considerable  length  of  time,  and  may  be  quite  independent 


CH.    IX.]  GLUCOSE.  161 

of  the  administration  of  carbohydrate  food.  The  con- 
dition is  known  as  diabetes  mellitus.  The  urine  is 
generally  much  increased  in  amount,  of  a  high  specific 
gravity,  and  pale  in  colour. 

The  classical  test  for  sugar  in  urine  is  Fehling's  (E'l.  67).  It  is  not  a 
reliable  test.  Not  only  is  Fehling's  solution  reduced  by  certain  con- 
stituents of  normal  urine,  such  as  urates  and  creatinine  ;  but  also  certain 
of  these  bodies,  notably  creatinine,  form  soluble  compounds  with  cuprous 
oxide,  and  thus  markedly  interfere  with  the  delicacy  of  the  test.  Further, 
glucose  is  destroyed  by  boiling  with  caustic  soda,  so  that  the  presence  of 
a  small  amount  of  sugar  may  escape  detection. 

Benedict's  test  (Ex.  63)  is  a  great  improvement.  Owing  to  the 
substitution  of  sodium  carbonate  for  sodium  hydroxide  it  is  not  reduced 
by  urates  or  creatinine,  and  it  does  not  destroy  small  quantities  of  sugar. 

Nylander's  test  (Ex.  70)  is  also  valuable.  The  reagent  is  not  reduced 
by  creatinine  or  uric  acid.  But  certain  substances  of  unknown  com- 
position that  are  occasionally  found  in  urine  cause  a  slight  reduction.  If 
a  negative  reaction  is  obtained  the  urine  may  be  regarded  as  free  from 
sugar  in  a  clinical  sense.  But  should  a  positive  reaction  be  obtained  this 
may  be  due  to  some  other  substance. 

The  osazone  test  serves  to  confirm  the  presence  of  sugar  in  doubtful 
cases,  and  especially  to  distinguish  between  glucose  on  the  one  hand  and 
lactose  and  peiitoses  on  the  other.  The  fermentation  test  is  also  valuable, 
especially  in  connection  with  the  recognition  of  lactose  and  glycuronic 
acid. 

If  proteins  are  present  they  must  be  removed  by  boiling  in  faintly 
acid  solution  before  performing  the  sugar  tests. 

290.  Benedict's  test.      To  5  c.c.  of  Benedict's  reagent    (see 
Ex.   68)  in  a  test  tube  add  eight  drops  of  the  urine.     Boil  vigor- 
ously for  two  minutes  and  allow  to  cool  spontaneously.     If  glucose 
is    present  the  entire   body   of   the   solution   will   be  filled  with  a 
precipitate  which  may  be  red,  yellow  or  green  in  colour,  depending 
on  the  amount  of  sugar. 

291.  Nylander's  test.     To  2  c.c.  of  Nylander's  reagent  (see 
Ex.  70)  in  a  test  tube   add    10   c.c.  of  urine  (free  from  protein). 
Boil  and  immerse  the   tube  in  a  beaker  of  boiling  water   for    5 
minutes.     The  presence  of  sugar  is  indicated  by  a  black  colouration 
or  precipitate  of  bismuth. 

NOTES  1. — Rusting  claims  that  the  addition  of  a  couple  of  drops  of  chloride 
of  platinum  (Pt  CM  increases  the  delicacy  of  the  test. 

M 


162  URINE.  [CH.    IX. 

2. — Bohmansonn  suggests  removing  urochrome  and  certaia  other  interfer- 
ing substances  by  the  following  method  :  Treat  10  c.c.  with  2  c.c.  of  25  per 
cent.  HC1  and  a  small  teaspoonful  of  animal  charcoal.  Shake  at  intervals  for 
five  minutes  and  then  filter.  Neutralise  the  clear,  colourless  filtrate  with 
caustic  soda. 

292.  Fehling's  test.     Boil  5   c.c.  of  Fehling's  solution  (see 
Ex.  67)  to  ascertain  whether  the  Rochelle  salt  has  been  decomposed 
into    reducing   substances.     If  no   reduction    occurs  add  an   equal 
volume  of  the  urine  (previously  freed  from  proteins)  and  boil  for  a 
short  time.     A  red  or  yellow  precipitate  indicates  the  presence  of 
glucose. 

NOTES  1. — This  test  is  satisfactory  for  the  demonstration  of  a  considerable 
quantity  of  glucose  (see  small  print  above). 

2. — Chloroform  must  not  be  used  as  a  preservative  if  this  test  be  used. 
Chloroform  is  converted  into  formic  acid,  a  reducing  substance, ^on  being  boiled 
with  sodium  hydroxide. 

293.  Phenylhydrazine  test.     Treat  10  c.c.  of  the  urine  with 
a  few  drops  of  lead  acetate   and  niter.     To  the  filtrate  add  5  or  6 
drops  of  strong  acetic  acid,  enough  phenylhydrazine  hydrochloride 
to  cover  a  sixpenny  piece,  and  twice  this  bulk  of  solid  sodium  acetate. 
Dissolve  by  the  aid  of  heat  and  filter.     Place  the  filtrate  in  a  tube 
and  immerse   this  in  a  boiling  water-bath  for  30   to  60  minutes. 
Turn  out  the  flame  and  allow  the  tube  to  cool  to  room  temperature 
without     removing     it    from    the    bath.       Examine    the    deposit 
microscopically  for  the  characteristic  crystals  of  phenylglucosazone. 
(See  Ex.  73.) 

NOTES  1. — In  doubtful,  cases  50  c.c.  of  urine  should  be  used,  with  a 
corresponding  increase  in  .the  amount  of  the  reagents.  The  precipitate  is 
collected  on  a  small  paper  and  dissolved  in  a  small  quantity  of  hot  alcohol.  The 
hot  alcoholic  solution  is  treated  with  boiling  water,  drop  by  drop,  till  a  turbidity 
is  produced.  The  solution  is  then  placed  in  a  boiling  water  bath  to  drive  off  the 
alcohol.  On  cooling  the  osazone  is  always  crystalline.  It  can  be  dried  and 
identified  by  taking  its  melting  point. 

294.  Cipollina's  test.     Place  5  drops  of  pure  phenylhydrazine 
(the  base)  in  a  test  tube.     Add  '5  c.c.  of  glacial  acetic  acid  and  4  c.c. 
of  urine.     Heat  the  mixture  over  a  low  flame,  shaking  continuously 
to  avoid   bumping,  and  keep  it  boiling  for   1  minute.     Add'4  to  5 
drops  of  40  per  cent,  sodium  hydroxide,  keeping  the  reaction  acid, 


CH.    IX.]  .  SUGARS.  163 

heat  for  a  moment  and  then  cool.     Crystals  of  the  osazone  usually 
form  at  once. 

295.  Fermentation  test.     Fill  a  test  tube  with  urine  and 
then  transfer  the  fluid  to  a  mortar.     Add  a  piece  of  washed  yeast 
about  the  size  of  a  bean  and  pound  it  up  with  the  urine.     Transfer 
the  mixture  to  the  test  tube  and  invert,  placing  the  open  end  under 
mercury  or  urine  contained  in  a  small  dish.     Clamp  the  tube  in 
position,  and  allow^  it  to  stand  for  at  least  eighteen  hours  in  a  warm 
place.     If  glucose  is  present  in  the  urine  there  is  an  accumulation 
of  gas  (CO2)  at  the  top  of  the  tube. 

NOTES  1. — Lactose,  pentoses  and  glycuronic  acids  are  not  fermented  by 
pure  yeast. 

2. — A  special  apparatus  called  Einhorn's  saccharometer  has  been  devised  to 
enable  the  test  to  be  applied  conveniently.  Also  the  volume  of  CO2  formed,  and 
the  percentage  of  glucose  present  can  be  roughly  determined  by  means  of  it. 

7.    Fructose   (laevulose). 

Fructose  occasionally  occurs  in  the  urine,  sometimes 
being  accompanied  by  glucose.  The  significance  of 
fructosuria  is  not  yet  clear. 

296.  Seliwanoff  s  test  (Borchardt's  modification).     To  a  few 
c.c.  of  urine  in  a  test  tube  add  an   equal  volume  of  25  per  cent, 
hydrochloric  acid  and  a  speck  or  two  of  resorcin.     Heat  to  boiling, 
cool  under  the  tap,  and  transfer  to  an  evaporating  dish.     Make  the 
reaction  alkaline  by  means  of  solid  sodium  hydroxide  and  return  it 
to  a  test  tube.     Add  3  c.c.  of  acetic  ether  (ethyl  acetate)  and  shake. 
A  yellow  colouration  in  the  acetic  ether  indicates  the  presence  of 
fructose. 

8.    Pentoses. 

Pentoses,  that  is  carbohydrates  with  5  carbon  atoms, 
appear  in  the  urine  in  three  conditions,  alimentary, 
persistent  or  true  pentosuria,  and  admixed  with  glucose 
in  cases  of  glycosuria. 

Alimentary  pentosuria  is  sometimes  seen  after  the 
iiigestion  of  considerable  quantities  of  certain  fruits,  as 


164  URINE.  [CH.    IX. 

prunes,  cherries,  grapes  and  plums.  The  sugar  found 
varies,  but  is  usually  r?-arabinose.  In  true  pentosuria  it 
is  dZ-arabinose.  Its  origin  and  significance  have  not  yet 
been  clearly  established. 

The  presence  of  pentoses  in  urine  is  indicated  when 
Nylander's  reaction  gives  a  grey  and  not  a  black  pre- 
cipitate :  when  Fehling's  test  shows  a  very  slow  reduction 
that  often  occurs  quite  suddenly  as  the  mixture  cools,  and 
when  the  fermentation  test  is  negative.  The  two  colour 
reactions  described  are  also  given  by  glycuronic  acid, 
which  can,  however,  be  demonstrated  by  Ex.  303. 

297.  Tollen's  test.     To  5  c.c.  of  urine  add  an  equal  volume 
of  strong  hydrochloric  acid  and  a  little  phloroglucin  (a  piece  about 
the  size  of  a  pea)  and  heat  the  mixture  on  a  boiling  water  bath. 
A  cherry -red  colour  develops  and  the  solution  shows  an  absorption 
band  between  D  and  E.     On  cooling  a  dark  precipitate  separates 
out.     On  dissolving  this  in  strong  alcohol,  the    alcoholic    solution 
shows  the  colour  and  absorption  band  of  the  original  mixture. 

298.  Bial's  orcin  test.     To  2  -  3  c.c.  of  urine  add  4  -  5  c.c.  of 
Bial's  reagent  and  heat  till  boiling  commences.     A  green  colour  or 
the    formation    of    a    green    precipitate    indicates    pentoses.      The 
solution  shows  two  absorption  bands,  one  in  the  red  between  B  and 
C  and  the  other  near  the  D  line. 

9.    Lactose. 

Lactose  is  found  in  the  urine  of  women  during 
pregnancy,  during  the  nursing  period,  and  soon  after 
weaning.  The  amount  in  the  urine  varies,  but  rarely 
exceeds  1  per  cent.  The  excretion  usually  reaches  its 
maximum  2  to  4  days  after  parturition. 

It  is  not  easy  to  demonstrate  the  presence  of  lactose 
in  urine  very  satisfactorily.  Barfoed's  test  is  not  applicable, 
oAving  to  the  fact  that  the  reagent  is  reduced  by  the 
constituents  of  normal  urine. 


CH.    IX.]  LACTOSE.  165 

The  osazone  cannot  be  isolated  with  any  certainty, 
owing  to  its  solubility.  Should  a  marked  reduction 
occur,  and  if  osazone  crystals  cannot  be  obtained,  the 
fermentation  test  should  be  applied,  using  pure  yeast  that 
has  been  tested  against  lactose.  If  this  be  negative,  then 
the  sugar  present  is  either  lactose  or  a  pentose.  Should 
the  tests  for  pentoses  yield  negative  results,  lactose  is 
indicated.  Its  presence  can  be  confirmed  by  obtaining 
crystals  of  mucic  acid,  which  is  yielded  only  by  lactose 
or  galactose. 

299.  Mucic  acid  test.  100  c.c.  of  the  urine  and  20c.c.  of 
pure  concentrated  nitric  acid  are  evaporated  in  a  wide  and  rather 
shallow  beaker  on  a  boiling  water  bath  in  a  fume  chamber.  The 
evaporation  is  continued  until  the  fluid  becomes  clear,  and  brown 
fumes  are  no  longer  evolved.  The  total  volume  is  then  about 
20  c.c.  Remove  the  beaker  from  the  bath  and  transfer  the 
contents  to  a  smaller  beaker,  washing  out  with  a  small  amount 
of  distilled  water.  Allow  to  stand  overnight  in  a  cool  place. 
The  formation  of  a  white  crystalline  mass  of  mucic  acid  indicates 
the  presence  of  lactose  in  the  urine.  Dilute  the  fluid,  collect 
the  crystals  on  a  small  filter  and  wash  with  cold  water. 
Microscopically  the  crystals  are  seen  to  be  very  pointed  prisms 
with  oblique  angles.  The  melting  point  is  213°- 215°  C.  It 
can  be  weighed  and  titrated  with  standard  alkalies,  its  equivalent 
weight  being  105. 

NOTE.— Mucic  acid  is  COOH.(CHOH)4COOH. 

10.     The  Acetone   bodies. 

The  acetone  bodies  found  in  urine  in  the  condition 
known  as  "  acidosis  "  are 

Acetone.     CH3.CO.CH3. 

Aceto-acetic  acid.     CH3.CO.CH2.COOH. 

/3-oxy-butyric  acid  CHa.CH(OH).CH.2.COOH. 

/3-oxy-butyric  acid  is  readily  oxidised  to  aceto-acetic 
acid,  and  this  is  converted  into  acetone  by  the  loss  of  C0.2. 


166  URINE.  [CH.    IX. 

The  two  acids  'are  never  found  in  urine  unaccom- 
panied by  acetone  :  but  acetone  may  be  present  without 
the  acids.  The  excretion  of  the  acetone  bodies  depends 
on  the  inability  of  the  tissues  to  oxidise  completely  the 
fatty  acids  generally  derived  from  the  fats,  but  sometimes 
from  certain  of  the  amino-acids  formed  in  the  metabolism 
of  proteins.  The  condition  that  usually  gives  rise  to 
acetonuria  or  acidosis  is  the  inability  of  the  tissues  to 
obtain  or  to  utilise  an  adequate  amount  of  glucose.  Thus 
these  acetone  bodies  are  excreted  in  starvation,  on  a  diet  of 
fats  with  a  limited  amount  of  protein,  in  certain  fevers, 
severe  anaemias,  and  after  phosphorus  poisoning,  and 
finally  in  diabetes  mellitus,  in  which  condition  the  tissues 
are  unable  to  utilise  the  glucose  provided.  ,. 

300.  Rothera's  test  for  acetone.    To  lOc.c.  of  the  urine 
add  an  excess  of  solid  ammonium  sulphate,  so  that  the  urine  is 
completely  saturated.     Then  add  two  or  three  drops  of  a. freshly 
prepared  5  per  cent,  solution  of  sodium  nitroprusside  and  2  or  3  c.c. 
of  concentrated  ammonia.     Mix  and  allow  to  stand  undisturbed  for 
at  least  thirty  minutes.     A  characteristic  permanganate  colouration, 
that  may   only  develop  above  the   layer  of   undissolved    crystals, 
indicates  the  presence  of  acetone. 

301.  Gunning's  iodoform  test  for  acetone.    To  200  c.c.  of 
urine,  add  a  few  drops  of  25  per  cent,  hydrochloric  acid  and  distil 
over  about  20  c.c.,  using  an  efficient  condenser.     To  5  c.c.  of  the 
distillate  add  five  to  ten  drops  of  10  per  cent,  ammonia  and  about 
five    drops    of    a    solution    of    1    part    of    iodine    and    2    parts    of 
ammonium  iodide  in   100  parts  of  water.     A  black  precipitate  of 
nitrogen    iodide    is    formed,    which   is   converted  on   standing   into 
iodoform.     Examine  the  sediment  microscopically  for  the  charac- 
teristic yellow  crystals  of  iodoform  (hexagonal  plates  and  rosettes). 

NOTES  1. — This  test  is  not  given  by  alcohol  or  aldehyde. 

2. — Aceto-acetic  acid  is  converted  into  acetone  on  boiling,  and  so  will  also 
give  the  test. 


CH.    IX.]  ACETO-ACETIC    ACID.  167 

302.  Gerhardt's  test  for  aceto-acetic  acid.    A.  To  5  c.c.  of 
the  urine  in  a  test-tube  add  ferric  chloride  solution,  drop  by  drop, 
till   no   further   precipitate  of  ferric  phosphate  is  formed.     Filter. 
To    the  filtrate  add  some  more  ferric   chloride.     A    Bordeaux-red 
colour  indicates  aceto-acetic  acid. 

NOTE. — A  similar  colour  is  given  by  a  large  number  of  substances,  such  as 
salicylic  acid,  and  the  bodies  excreted  after  the  administration  of  aspirin, 
antipyrin,  thallin,  etc.  The  majority  of  these  substances  are  not  destroyed  by 
boiling,  whereas  aceto-acetic  acid  is  converted  into  acetone. 

B.  If  A  is  positive  shake  50  c.c.  of  urine  and  3  drops  of 
strong  sulphuric  acid  with  ether.  Pipette  off  the  ether  and  treat 
it  with  very  dilute  ferric  chloride.  The  lower  layer  becomes 
coloured  violet.  Add  more  ferric  chloride.  The  colour  changes 
to  a  Bordeaux-red. 

NOTE.  —  It  is  advisable  to  shake  the  acidified  urine  first  with  chloroform  or 
benzene,  to  extract  salicylic  acid. 

11.     Glycuronic  Acid. 

Glycuronic  acid,  CHO.(CHOH)1.COOH,  is  not  found 
free  in  the  urine.  It  is  found  conjugated  with  certain 
drugs,  or  with  substances  formed  from  these  in  the  body. 
These  conjugated  glycuronates  are  excreted  after  ad- 
ministration of  chloral,  camphor,  naphthol,  menthol, 
phenol,  morphine,  oil  of  turpentine,  antipyrin,  etc.  The 
free  and  conjugated  acids  are  reducing  substances,  but 
are  not  fermentable.  They  give  the  reactions  for  the 
pentoses,  but  can  be  distinguished  by  the  test  given  below. 

303.  Tollen's  test  for  glycuronates.    To  5  c.c.  of  the  urine 
in  a  rather  wide  test-tube  add  '5  to  1  c.c.  of  a  1  per  cent,  solution  of 
naphthoresorcin  in  alcohol  and  5  to  6  c.c.  of  strong  hydrochloric 
acid.     Heat  slowly  to  boiling  point  and  keep  boiling  for  1  minute, 
shaking  the  tube  the  whole  time.     Set  the  tube  aside  for  4  minutes, 
then  cool  under  the  tap  and  shake  with  an  equal  volume  of  ether. 
The  ether  is  coloured  violet  to  red,  and  when  examined  spectro- 
scopically  shows  two  bands,  one  on  the  D  line,  and  one  to  the  right 
of  it. 


168  URINE.  [CH.    IX. 

12.    Indican. 

Indican   is   the  potassium  salt  of  indoxyl  sulphuric 
acid,  and  is  thus  one  of  the  ethereal  sulphates  (see  p.  135). 

Indoxyl  is 

CH 


HC  C C.OH 

i      «     I 

HC  C  C.H 

\/\/ 

CH        NH. 

Indican  is 

CH 


HC          -    C C.O.SOsK 

!    I 

HC  C  CH 

\/  \/ 

CH         NH. 

Indoxyl  arises  from  the  bacterial  decomposition  of 
tryptophane  in  the  intestine,  thus  differing  from  the 
other  ethereal  sulphates  which  are  normal  tissue  meta- 
bolites (see  p.  135).  The  excretion  of  indican  is  of 
importance  as  a  measure  of  the  amount  of  putrefaction 
occurring,  generally  in  the  intestine,  but  sometimes  in  a 
large  abscess. 

304.  Jaffe's  test.  Treat  5  c.c.  of  urine  with  a  rather  larger 
volume  of  concentrated  hydrochloric  acid  and  about  2  c.c.  of 
chloroform.  Add  a  single  drop  of  3  per  cent,  potassium  chlorate 
and  shake.  Allow  the  chloroform  to  settle  and  examine  its  colour. 
If  it  be  blue,  indican  is  present.  If  not,  add  another  drop  of  the 
chlorate  and  shake  again.  If  no  blue  colour  be  found  in  the 
chloroform,  indican  is  absent. 


CH.    IX.]  URINARY     SEDIMENTS.  169 

305.  Lavelle's  test.  Treat  10  c.c.  of  urine  with  2-3  c.c.  of 
Obermayer's  reagent.  Add  2-3  c.c.  of  concentrated  sulphuric  acid 
slowly,  cooling  under  the  tap  during  the  addition.  Add  2-3  c.c.  of 
chloroform  and  shake.  A  blue  colouration  in  the  chloroform 
indicates  the  presence  of  indican. 

NOTES. — 1.  In  these  reactions  the  strong  acids  hydrolyse  indoxyl  sulphuric 
acid  to  free  indoxyl  and  sulphuric  acid.  The  oxidising  reagents  oxidise  the 
indoxyl  to  indigo  blue,  which  is  soluble  in  chloroform. 

2.  Obermayer's  reagent  is  prepared  by  dissolving  4  gm.  ferric  chloride 
in  1  litre  of  concentrated  hydrochloric  acid. 

L.     Urinary   Sediments. 

For  the  proper  examination  of  these  substances  a 
hand  centrifuge  is  desirable.  The  sediment  obtained 
should  be  examined  microscopically,  and  chemically  if 
necessary. 

The  sediments  obtained  are  either  organised  or 
unorganised.  Organised  sediments  consist  of  casts  of  the 
renal  tubules,  epithelial  cells  from  different  parts  of  the 
urinary  tract,  pus,  blood  cells,  spermatozoa,  parasites,  etc. 
It  is  not  thought  advantageous  to  describe  them  in  this 
book. 

Unorganised  sediments  vary  with  the  reaction  of  the 
urine.  The  more  common  varieties  are  given  below. 

In   acid    urine. 

Uric  acid:  light  yellow  to  dark  reddish-brown  in 
colour.  Crystalline  form  very  varied :  rhombic  prisms, 
wedges,  rosettes,  dumb-bells,  whetstones,  butcher's  trays, 
etc.  Soluble  in  sodium  hydroxide  and  reprecipitated  by 
hydrochloric  acid. 

Urates :  pinkish,  soluble  on  warming,  sometimes 
amorphous,  sometimes  crystalline,  as  "  thorn-apples,"  fan- 
shaped  clusters  of  prismatic  needles. 


170  URINE.  [CH.    IX. 

Calcium  oxalate:  octahedra,  with  an  envelope-like 
appearance  (squares  crossed  by  two  diagonals) ;  also  in 
dumb-bells.  Insoluble  in  acetic  acid,  easily  soluble  in 
hydrochloric  acid. 

Calcium  hydrogen  phosphates  (stellar  phosphates):  in 
rosettes  of  prisms  and  in  dumb-bells.  Rather  rare. 

Cystine:  colourless  hexagonal  plates,  soluble  in 
ammonia,  insoluble  in  acetic  acid.  Very  rare. 

In  alkaline   urine. 

Ammonium  magnesium  phosphate  (triple  phosphate) : 
colourless  prisms  ("  coffin-lids  "  and  "  knife-rests ")  or 
feathery  stars.  Easily  soluble  in  acetic  acid. 

Alkaline  earthy  phosphates  of  calcium  and  magnesium  : 
amorphous.  Insoluble  on  warming  and  in  alkalies, 
soluble  in  acetic  acid. 

Calcium  hydrogen  phosphate :  see  above. 

Calcium  carbonate:  dumb-bells  or  spheres  with 
radiating  structure 

Ammonium  urate:  yellow,  or  brownish  amorphous 
masses,  or  shewing  "  thorn-apple  "  crystals.  Soluble  on 
warming. 


CHAPTER  X. 
THE  QUANTITATIVE  ANALYSIS  OF  URINE. 

To  determine  the  nature  of  the  metabolic  processes 
in  the  body  a  sample  of  the  measured  24  hours'  urine 
must  be  analysed.  In  taking  the  24  hours'  urine  it  is 
best  to  finish  with  that  voided  after  the  night's  rest. 
The  total  collected  during  the  24  hours  is  mixed  and 
carefully  measured.  The  analyses  should  be  performed 
as  soon  as  possible,  owing  to  the  risk  of  bacterial  decom- 
position of  certain  of  the  constituents.  Should  it  be 
necessary  to  postpone  the  analyses  an  antiseptic  should 
be  added.  Toluol  or  thymol  are  the  best  to  use  (but 
see  Ex.  278,  note  7).  Chloroform  must  not  be  used  in 
any  case,  since  it  is  decomposed  by  alkalies  and  has  a 
marked  effect  on  certain  processes. 

The  analyses  performed  will  vary  with  the  nature 
of  the  case  that  is  being  investigated,  .and  the  time 
and  apparatus  at  the  disposal  of  the  analyst.  It  is 
of  the  utmost  importance  for  the  student  to  acquire 
skill  in  the  conduction  of  a  complete  analysis,  and 
in  this  connection  particular  attention  is  directed  to 
Folin's  micro-chemical  methods,  based  on  colorimetric 
comparison,  that  are  described  below.  They  enable  a 
complete  analysis  of  the  nitrogenous  constituents  of  a 
sample  of  urine  to  be  made  in  a  few  hours  with  a  very 
small  amount  of  special  apparatus  beyond  a  good  suction 
pump  and  a  reliable  colorimeter,  preferably  Dubosq's. 


172 


ANALYSIS     OF     URINE. 


[CH.    X. 


Since  the  fumes  arising  from  the  incineration  of 
urine  by  boiling  sulphuric  acid  are  extremely  irritating, 
that  operation  should  be  conducted  in  a  fume  chamber 
or  under  a  hood.  But  these  can  be  dispensed  with  by 
use  of  the  special  fume-absorber  devised  by  Folin  and 
illustrated  in  Fig.  6.*  A  is  a  bulb  (1^  inches  in  diameter) 
blown  into  a  piece  of  fths  Jena  tubing.  The  lower 


TO  PUMP- 


NaOH 


Fig.  6.     Folin' s  fume-absorber. 


*  The  apparatus  described  in  this  book  can  now  be  obtained  from 
Messrs.  J.  Griffin  and  Sons,  or  from  Messrs.  Baird  and  Tatlock,  and 
will  be  listed  in  their  next  catalogues. 


CH.    X.J  TOTAL     NITROGEN.  173 

(Mid  has  blown  into  it  a  piece  of  narrow  tubing  (C) 
2£  inches  in  length.  The  bulb  rests  on  the  neck  of 
the  flask  or  test-tube  in  which  the  incineration  is 
conducted. 

To  the  upper  end  of  the  tube  is  fixed  a  piece  of 
narrow  tubing  which  is  bent  at  a  convenient  angle, 
and  which  slips  into  a  slightly  longer  tube  connected 
to  a  good  suction  pump.  The  fumes  are  carried  over 
by  the  air  current  into  the  pump,  a  wash  bottle  containing 
caustic  ,soda  being  interposed  to  prevent  damage.  The 
condensation  water  collects  in  the  pocket  C  and  can 
be  removed  by  inverting  the  fume-absorber  at  the  end 
of  the  experiment.  The  removal  of  this  condensation 
water  materially  hastens  the  incineration. 

One  good  pump  suffices  to  carry  off  the  fumes  from 
three  or  four  incinerations  simultaneously. 

By  inverting  a  funnel  over  an  evaporating  basin,  and 
arranging  the  apparatus  so  that  the  end  of  the  funnel 
fits  loosely  into  the  neck  of  the  absorber,  the  fumes 
from  boiling  nitric  acid  can  be  carried  off. 

306.  The  estimation  of  total  nitrogen  by  Kjeldahl's 
method. 

Principle.  The  nitrogenous  compounds  in  5  c.c.  of  urine 
are  converted  into  ammonium  sulphate  by  boiling  with  sulphuric 
acid,  copper  sulphate  being  added  to  aid  the  oxidation,  and  potassium 
sulphate  to  raise  the  boiling  point.  The  mixture  is  diluted  with 
water,  made  alkaline  by  the  addition  of  sodium  hydroxide 
and  the  ammonia  distilled  into  a  measured  amount  of  standard 
acid.  The  amount  of  this  neutralised  by  the  ammonia  is  found 
by  subsequent  titration  with  standard  alkali.  Knowing  the  amount 
of  ammonia  formed  from  5  c.c.  of  urine,  the  percentage  of 


174 


ANALYSIS     OF     URINE. 


[CH.    X. 


Method    of   Analysis. 


nitrogen  can  be  readily  calculated. 
Into  a  clean,  dry,  round-bottomed 
flask  of  Jena  glass  A  (500  c.c. 
capacity,  with  a  narrow  neck  8  inches 
in  length)  place  10  gms.  potassium 
sulphate,  2  c.c.  of  saturated  copper 
sulphate  solution,  5  c.c.  of  urine 
(accurately  measured)  and  10  c.c. 
of  concentrated  sulphuric  acid,  free 
from  nitrogen.  Place  the  flask  in 
the  fume-chamber  (or  use  the  fume- 
absorber,  described  on  page  172),  and 
heat  by  means  of  a  low  flame  for 
10-15  minutes,  then  boil  briskly 
for  45  minutes.  When  cool  add 
250  c.c.  of  ammonia-free  distilled 
water,  about  0*5  gm.  of  powdered 
pumice  and  cool  under  the  tap. 
Into  an  Erlenmeyer  flask,  E,  of 

about  400  c.c.  capacity,  place  25  c.c. 

N 
of  —    solution  of  sulphuric  acid. 


Fig.  7.— Apparatus  for  Kjeldahl's  This    flask    is   then    placed    on 

an    adjustable    stand,    so    arranged 

that  the  lower  end  of  the  tube  D  dips  below  the  surface  of  the 
acid  in  E.  The  bulb  in  D  is  to  decrease  the  risk  of  the  acid 
in  E  being  sucked  back  by  a  sudden  cooling  of  A  during  the 
distillation.  D  is  connected  to  a  condenser  C.  The  best  pattern 
is  Davies',  which  is  shewn  in  fig.  7. 

To  the  flask  A  add  35  c.c.  of  40  per  cent,  sodium  hydroxide, 
pouring  it  down  the  neck  and  wall  of  the  flask  so  as  to  form  a 
bottom  layer;  loss  of  ammonia  is  thus  prevented. 

Fit  the  glass  tube  B  into  the  neck  of  A  by  means  of  a 
well-fitting  rubber  stopper.  The  special  bulb  on  B  is  to 
prevent  any  of  the  alkaline  fluid  bumping  over  into  the  distillate. 


CH.    X.]  TOTAL    NITROGEN.  175 

Mix  the  contents  of  A  by  shaking  and  immediately  connect 
up  13  with  C  by  means  of  another  well-fitting  rubber  stopper. 
Heat  the  mixture  in  A  to  boiling  by  means  of  a  free  flame 
from  a  Bunsen  burner  provided  with  a  rose-jet.  Allow  the 
fluid  to  boil  till  at  least  half  the  total  volume  of  fluid  has 
distilled  over,  lowering  E  from  time  to  time,  so  that  D  does 
not  dip  too  far  under  the  acid.  Finally,  lower  E  so  that  the 
tube  no  longer  dips  under  the  surface  and  continue  the  boiling 
for  another  minute  or  two  to  wash  down  any  of  the  standard 
acid  that  may  have  been  sucked  up  into  the  tube  or  bulb. 
Wash  down  the  exterior  of  the  lower  end  of  D  with  a  jet  of 
distilled  water,  allowing  the  washings  to  run  into  E. 

To   the   fluid  thus  obtained  add  a  drop  of    a  dilute   solution 

of    neutral   red    (the   best  indicator),   methyl   orange,  cochineal   or 

N 
congo  red  and  titrate  with  —  sodium  hydroxide. 

Calculation  of  results.     Example  : 

N  N 

164  c.c.  of  —  =  8-2  c.c.  of  —  NaOH  found  necessary. 

N 
.'•  25  —  8'2  =  16'8  c.c.  of  —  acid  were  neutralised  by  ammonia 

from  5  c.c.  of  urine. 

1  c.c.  of  -  acid  =  -0028  gm.  N. 

/.  16-8  c.c =  -04704  gm.  N. 

So  N  per  cent.  =  -04704  X  20  =  -9408  gm. 

307.  The  estimation  of  total  nitrogen  by  Folin's  micro- 
chemical  method. 

Principle.  A  small  volume  of  urine  is  decomposed  by  sul- 
phuric acid  as  in  Kjeldahl's  method.  The  ammonia  is  drawn  over 
into  acid  and  the  solution  treated  with  Nessler's  reagent.  The 
amount  of  ammonia  is  determined  colorimetrically  by  comparison 
with  a  standard  solution  of  ammonium  sulphate  simultaneously 
Nesslerised. 


176 


ANALYSIS     OF     URINE. 


[CH.    X. 


Incineration.  Measure  5  c.c.  of  urine  into  a  50  c.c.  flask  if 
the  specific  gravity  be  over  1018,  or  into  a  25  c.c.  flask  if  the  sp.  gr. 
be  less  than  1018.  (The  dilution  must  be  such  that  1  c.c.  of  the 
diluted  urine  contains  between  0'75  and  1'5  mgms.  N.)  Fill  the  flask 
to  the  mark  with  distilled  water  and  invert  it  a  few  times  to  secure 
thorough  mixing.  Measure  1  c.c.  of  the  diluted  urine,  by  means  of 
an  accurate  pipette,*  into  a  large  test  tube  of  Jena  glass  (20  to 


AIR 
TROM 

WASHBOTTLE: 


SUCTION 


Fig.  8.     Apparatus  for  Folin's  micro-chemical  methods.f 

A.  Jena  test  tube,  20  to  25  mm.  by  200  mm. 

B.  Sheet  of  rubber,  about  2  mm.  thick,  cut  from  a  2-holed  rubber 

stopper.     It  fits  loosely  into  A,  and  has  a  small  groove  cut  at 
the  side. 

C.  Made  from  a  broken  5  c.c.  pipette. 

D.  100  c.c.  measuring  flask  with  wide  neck  to  take  a  2-holed  rubber 

stopper. 

E.  Tube  sealed  at  lower  end  and  holes  bored  in  it  by  means  of  a  hot 

platinum  wire.     It  prevents  loss  of  ammonia. 

To  prevent  any  ammonia  from  the  room  entering  the  apparatus 
the  air  is  passed  through  a  wash  bottle  containing  sulphuric 
acid  before  it  enters  A. 

*   Ostwald's  pipettes  are  suitable. 

The  various  parts  of  the  apparatus  can  be  obtained  from  Messrs.  Baird  and 
Tatlock,  and  from  Messrs.  J.  Griffin  and  Sons. 


CH.    X.]  TOTAL    NITROGEN.  177 

25  mm.  by  200  mm.)  Add  1  c.c.  of  pure  sulphuric  acid,  1  gram  of 
potassium  sulphate,  1  drop  of  5  per  cent,  copper  sulphate  and  a 
small,  clean  quartz  pebble,  or  a  small  fragment  of  granulated  zinc 
(to  prevent  bumping).  Boil  over  a  micro-burner  for  about  ten 
minutes,  either  in  the  fume  chamber  or  use  the  fume-absorber 
described  on  page  172.  Allow  to  cool  for  about  three  minutes  and 
then  add  about  6  c.c.  of  water,  at  first  a  few  drops  at  a  time,  then 
more  rapidly  so  as  to  prevent  the  mixture  solidifying. 

Distillation  of  the  Ammonia.  Use  the  apparatus  shewn  in 
Fig.  8.  Transfer  3  c.c.  of  a  saturated  solution  of  sodium  hydroxide 

to    the  mixture  in  the  test-tube  A,  and  aspirate  the  ammonia  into 

N 
2  c.c.  of  —   hydrochloric  aid  and  about  20  c.c.  of  distilled  water 

contained  in  the  special  100  c.c.  measuring  flask  D.  The  air 
current  may  be  moderate  for  the  first  two  minutes  but  for  the  next 
eight  minutes  it  should  be  as  rapid  as  possible.  Disconnect  and 
dilute  the  contents  to  about  60  c.c.,  washing  the  tube  E  with  some 
of  the  water  added. 

Preparation  of  the  Nesslerisefi  solutions.  In  another  100  c.c. 
measuring  flask  place  5  c.c.  of  standard  ammonium  sulphate 
solution,  containing  1  mgm.  of  nitrogen,  and  dilute  it  to  60  c.c. 
To  each  flask  add  3  c.c.  of  a  cold  saturated  solution  of  Rochelle 
salt  (to  prevent  the  formation  of  a  cloud  on  adding  Nessler's 
solution).  Nesslerise  both  solutions  as  nearly  as  possible  at  the 
same  time  with  5  c.c.  of  Nessler's  reagent  diluted  immediately 
beforehand  with  25  c.c.  of  water.  Fill  both  flasks  to  the  mark 
with  water  and  mix. 

Determination  of  the  depth  of  colour.  This  is  done  by  means 
of  a  Dubosq  colorimeter.  (See  Fig.  11,  p.  191.)  In  one  of  the 
chambers  B  place  some  of  the  unknown  solution,  in  the  other  some 
of  the  standard  ammonia  solution.  Place  the  tube  D  of  the 
standard  at  a  certain  depth  (20  mm.  is  usually  the  best)  and  adjust 
the  other  tube  until  the  colours  match.  Several  readings  should  be 
taken,  moving  the  unknown  from  below  and  from  above. 

N 


178  ANALYSIS     OF     URINE.  [CH.    X. 

Calculation  of  results.     Example  : 

Height   of  standard   _        20  mm. 
Height  of  unknown         21 '3  mm. 

20 
So  the  1    c.c  of  fluid  taken  contains  ^y—    ==  0'94  mgm. 

nitrogen. 

Urine  was  diluted  1  in  10. 
So  100  c.c.  of  urine  contains  0'94  gm.  nitrogen. 

Preparation  of  the  standard  solution  of  ammonium  sulphate. 
Pure  ammonium  sulphate  is  decomposed  by  means  of  caustic  soda 
and  the  ammonia  passed  into  pure  sulphuric  acid  by  means  of  the 
air  current.  When  all  the  acid  has  been  neutralised,  the  solution 
is  partially  evaporated  and  the  salt  precipitated  by  alcohol.  It  is 
redissolved  in  water,  reprecipitated  by  alcohol,  and  dried  in  a 
desiccator  over  sulphuric  acid. 

9*4285  gm.  of  the  ammonium  sulphate  are  dissolved  in  water 
and  the  volume  made  up  to  1  litre.  (Stock  solution). 

100  c.c.  of  the  stock  solution  are  diluted  to  form  1  litre 
(Standard  solution). 

5  c.c.  of  the  standard  solution  contain  1  mg.  nitrogen. 
Preparation  of  Nessler's  reagent. 

Dissolve  62'5  gm.  of  potassium  iodide  in  about  250  c.c.  of 
distilled  water,  set  aside  a  few  c.c.  and  add  gradually  to  the  larger 
part  a  cold  saturated  solution  of  mercuric  chloride  (of  which  about 
500  c.c.  will  be  required)  until  a  faint  permanent  precipitate  is 
produced.  Add  the  reserve  portion  of  the  potassium  iodide  and 
then  mercuric  chloride  very  gradually  till  a  slight  permanent 
precipitate  is  again  formed. 

Dissolve  150  gm.  of  solid  potassium  hydroxide  in  150  c.c.  of 
distilled  water,  allow  the  solution  to  cool  and  add  it  gradually  to 
the  above  solution  and  make  the  volume  up  to  1  litre.  Allow  to 
settle,  decant  the  clear  liquid  into  another  bottle  and  keep  in  the 
dark.  The  reagent  improves  on  keeping. 


CH.    X.J  AMMONIA.  179 

308.     The  estimation  of  ammonia  by  Folin's  method. 


E. 


AIR 


1 


T 


ABC 

Fig.  9.     Folin's  apparatus  for  estimating  ammonia, 

A.  Wash  bottle  containing  acid. 

B.  Tall  aerometer  cylinder  containing  urine. 

C.  Bottle  containing  standard  acid. 

D.  Calcium  chloride  tube,  loosely  packed  with  cotton  wool,  to  prevent 

any  sodium  carbonate  being  carried  over  into  C. 

Folin's  absorption  tube,  to  bring  the  air  into  intimate  contact  with 
the  acid. 


Use  the  apparatus  shown  in  Fig.  9*. 

N 
Into  C  measure  20  c.c.  of  —  sulphuric  acid  and  two  drops  of 

a  dilute  solution  of  methyl  red,  or  Alizarin  red. 

Into  B  measure  25  c.c.  of  urine,  add  10  c.c.  of  kerosene  oil  (to 
prevent  foaming)  and  one  gram  of  anhydrous  sodium  carbonate. 
Connect  up  the  apparatus  at  once,  and  draw  air  through  for  two 
hours. 


*  The  parts  of  the  apparatus  can  be  obtained  from  Messrs.  J.  Griffin  and  Sons, 
or  Messrs.  Baird  and  Tatlock. 


180  ANALYSIS    OF     URINE.  [CH.   X. 

Disconnect  the  apparatus,  wash  the  tube  E  with  distilled  water 

N 
into  C,  and  titrate  with  —  sodium  hydroxide. 

Calculation.     Subtract  the  number  of  c.c.  of  sodium  hydroxide 

N 
from  20.     The  result  is  the  number  of  c.c  of  —  sulphuric  acid 

neutralised  by  the  ammonia  in  25  c.c.  of  urine. 

N 
1  c.c.  of  —  acid  —  -0017  gm.  of  ammonia. 

=  -0014  gm.  of  ammonia  nitrogen. 

309.  The  estimation  of  ammonia  by  Folin's  micro- 
chemical  method. 

Use  the  apparatus  sketched  in  Fig.  8,  p.  176. 

Into  the  test  tube  A  measure  1  to  5  c.c.  of  urine,  so  that  0'75 
to  1-5  mgms.  of  ammonia-nitrogen  are  dealt  with.  For  normal 
urine  2  c.c.  are  usually  about  right.  With  diabetic  urine,  even 
1  c.c.  may  be  too  much,  and  the  urine  must  be  previously  diluted. 

Add  water,  if  necessary,  to  bring  the  volume  to  about  5  c.c. 
Add  a  few  drops  of  a  solution  containing  10  per  cent,  of  potassium 
carbonate  and  15  per  cent,  of  potassium  oxalate.  Also  a  few  drops 
of  kerosene  or  heavy,  crude  machine  oil  (to  prevent  foaming). 

N 
Measure    2.    c.c.    of  —  hydrochloric    acid    into    the    100    c.c. 

graduated  flask  D,  add  about  20  c.c.  of  distilled  water,  connect  up 
the  apparatus  and  pass  a  strong  current  of  air  through  for  15 
minutes.  Nesslerise  as  described  in  Ex.  307,  and  compare  with 
1  mgm.  of  nitrogen  obtained  from  the  solution  of  standard  ammo- 
nium sulphate,  similarly  and  simultaneously  Nesslerized. 

Calculation.  The  number  of  mgms.  of  ammonia-nitrogen  in 
the  volume  of  urine  taken  are  readily  calculated  as  in  Ex.  *207, 
and  so  the  number  of  grams  per  100  c.c.  The  amount  of  ammonia 
is  obtained  from  this  by  multiplying  by  —  =  1*214.« 


CH.    X.]  AMMONIA.  181 

310.  The  estimation  of  ammonia  by  the  formaldehyde 
method. 

Principle.  When  neutral  solutions  of  ammonium  salts  are 
treated  with  formaldehyde,  combination  occurs  between  the  ammonia 
and  the  formaldehyde,  hexamethylene  tetramine  (urotropin)  being 
formed.  A  corresponding  amount  of  acid  is  liberated  from  the 
ammonium  salt,  and  this  can  be  titrated  with  standard  alkali. 

4  NH4C1  +  6  CH20  =  N4(CH2)6  +  6  H2O  +  4  HC1. 

Formaldehyde.     Urotropin. 

The  objection  to  the  method  is  that  amino-acids  react  in  a 
similar  manner,  so  that  the  result  obtained  is  ammonia  +  amino- 
acids.  However,  it  is  of  considerable  value  as  a  rapid  clinical 
method  of  determination. 

Preparation  of  formaldehyde  solution.  5  c.c.  of  commercial 
formalin  (40  per  cent,  formaldehyde)  are  diluted  with  5  c.c.  of 
water,  treated  with  a  drop  of  phenolphthalein  and  neutralised  with 

—  sodium  hydroxide. 

N 
Method  of  estimation.     Neutralise  25  c.c.   of  urine  with  — 

sodium  hydroxide  exactly  as  described  on  page  193.     To  the  mixture 

thus  obtained  add  the  whole  of  the  formaldehyde  solution.     Read 

N 
the  burette  and  run  in  more  —sodium  hydroxide  until  the  same  point 

is  obtained  as  before. 

N 
Calculation  of  results.     1  c.c.  of  —  sodium  hydroxide 

--=  -0017  gm.  NH3 

=  '0014  gm.  of  ammonia-nitrogen. 

311.  The  estimation  of  urea  by  Benedict's  method. 

Principle.  Urine  is  treated  with  potassium  bisulphate  and  zinc 
sulphate  and  heated  to  165°C.  for  one  hour.  The  urea  is  thus 
hydrolysed  to  ammonium  compounds  which  are  retained  by  the 
acid  mixture.  The  fluid  is  diluted,  made  alkaline  with  sodium 
-carbonate,  and  the  ammonia  distilled  into  standard  acid.  The 


182  ANALYSIS     OF     URINE.  [CH.    X. 

amount  of  this  neutralised  by  the  ammonia  formed  is  determined  by 
titration  with  standard  alkali.  The  ammonia  nitrogen  of  the  urine 
must  be  previously  determined. 

Method.  5  c.c.  of  urine  are  measured  into  a  wide  Jena  test-tube 
(200  X  25mm.)  and  treated  with  about  3  grams  of  potassium 
bisulphate  and  1  to  2  grams,  of  zinc  sulphate.  A  little  powdered 
pumice  and  a  bit  of  paraffin  are  introduced  to  minimise  frothing 
and  spattering,  and  the  mixture  boiled  practically  to  dryness,  either 
over  a  small  free  flame  or,  more  conveniently,  by  floating  the  tube  in 
a  bath  of  sulphuric  acid  kept  at  about  130°  C.  A  convenient  bath  is 
a  tall-form  Jena  glass,  or,  preferably,  porcelain  beaker  of  about 
800- 1,000  c.c.  capacity,  two-thirds  full  of  sulphuric  acid. 

The  tube  is  then  immersed  for  at  least  three-fourths  of  its 
length  in  the  sulphuric  bath.  This  can  be  done  by  clamping  the 
tube  to  the  edge  of  the  bath.  Raise  the  temperature  of  the  bath  to 
162°  -  165°  C.  and  maintain  it  there  for  one  hour.  Remove  the  tube 
and  allow  it  to  cool  somewhat.  Wash  off  the  acid  under  the  tap. 
Wash  the  contents  by  means  of  hot  water  quantitatively  into 
a  500  c.c.  Jena  flask  (A,  fig.  7,  p.  174).  The  volume  of  the  fluid 
in  the  flask  should  be  about  350  c.c. 

Fit  up  the  apparatus  as  used  for  Kjeldahl's  method,  placing 

N 
25  c.c.    of   —   sulphuric   acid  in   D.     To  A  add  about   25  c.c.   of 

a  saturated  solution  of  sodium  carbonate.  Connect  up  the  appar- 
atus and  distil  for  about  forty  minutes,  till  about  one-half  of  the 
fluid  has  passed  over.  Boil  the  fluid  in  D  to  remove  excess  of 
CO2,  cool  and  titrate  with  N/10  sodium  hydroxide,  using  methyl 
red,  cochineal  or  methyl  orange  as  an  indicator. 

Calculation  of  results.     Example  : 

Ammonia-nitrogen   of   25  c.c.   urine  was  previously   found  to- 

N  N 

correspond  to  10  c.c.  —  H2SO4  =  5  c.c.— H2SO4. 

N 

So  ammonia-nitrogen  of  5  c.c.  urine  =  1  c.c.— H2SOi. 

j 


CH.    X.]  UREA.  183 

In   this   exercise    16  c.c.  of  — NaOH,  i.e.  8  c.c.  of  yNaOH 

N 
neutralised  the  25  c.c.— H2SO4. 

N 
So  amount  of  — H^SCX   neutalised   by  urea  and  ammonia  was 

25-8  =  17  c.c. 

Amount  neutralised  by  urea  alone  was  therefore  17  —  1  =  16  c.c. 
1  c.c.  of  yH.2SO4  =  -0028  gm.  N  =  '006  gm.  urea. 

So  Urea-N  in  5  c.c.  =  16  X  -0028  =  -0045  gm. 
Urea  in  5  c.c.         =  16  X  -006    =  '096  gm. 
Urea  per  cent.       =  -096  x  20    =  1-92. 

312.  The  estimation  of  urea  by  Folin's  microchemical 
method. 

Principle.  Urine  is  treated  with  potassium  acetate  and 
acetic  acid  and  boiled.  The  boiling  point  of  the  mixture  is  about 
155°  C.,  and  at  this  temperature  the  urea  is  rapidly  hydrolysed  to  CO2 
and  ammonia,  which  is  retained  as  ammonium  acetate.  Caustic 
potash  is  added,  and  the  ammonia  aspirated  into  acid.  This 
solution  is  Nesslerised  and  the  colour  compared  with  that  of 
a  standard  solution  of  ammonium  sulphate  similarly  and  simul- 
taneously Nesslerised. 

Method.  The  urine  must  be  diluted  so  that  1  c.c.  contains 
0*75  to  1'5  mgms.  of  urea  nitrogen.  Usually  1  in  10  is  about 
correct.  In  a  large  dry  Jena  test  tube  (A,  fig.  8,  p.  176)  place  7  grams 
of  dry  potassium  acetate,  1  c.c.  of  50  per  cent,  acetic  acid,  a  small 
fragment  of  granulated  zinc  (to  prevent  bumping),  and  a  temperature 
indicator  (see  below).  To  the  tube  transfer  1  c.c.  of  the  diluted 
urine  by  means  of  an  accurate  pipette.  The  test  tube  is  then 
closed  by  means  of  a  rubber  stopper  carrying  an  empty  narrow 
"calcium  chloride  tube,"  without  bulb  (25cm.  by  1*5 cm.)  The 
test  tube  is  held  by  a  clamp,  so  that  it  can  be  readily  raised 
or  lowered.  Heat  is  applied  by  means  of  a  microburner,  which  is 


184  ANALYSIS     OF     URINE.  [cH.    X. 

shielded  from  air  currents  by  means  of  a  chimney  or  a  bottomless 
beaker.     The  flame  should  be  about  0*5  cm.  long. 

The  acetate  dissolves  and  the  mixture  begins  to  boil.  The 
temperature  indicator  should  show  that  the  temperature  has  reached 
153°C.  to  160°C.  Boiling  is  continued  for  ten  minutes  after  this  tem- 
perature has  been  attained.  (Th e  temperature  must  not  reach  1 6  2°  C . , 
at  which  point  the  acetate  cakes  and  solidifies.)  Remove  the 
apparatus  from  the  flame  and  dilute  the  contents  with  5  c.c.  of 
water,  adding  it  from  a  pipette  through  the  calcium  chloride  tube 
so  as  to  rinse  the  sides  of  the  tube  and  the  bottom  of  the  rubber 
stopper  from  traces  of  ammonium  acetate  which  may  be  there,  add 
2  c.c.  of  saturated  sodium  hydroxide  solution  and  aspirate  the 
ammonia  into  acid  exactly  as  described  on  p.  179.  Estimate  the 
nitrogen  colorimetrically  against  1  mgm.  of  nitrogen  'as  described 
above. 

Temperature  indicator.  This  consists  of  powdered  chloride- 
iodide  of  mercury  (Hg  IC1)  enclosed  in  sealed  tubes  10  to  15  mm.  in 
length  and  not  over  1  mm.  in  diameter.  The  salt  is  bright  red  at 
ordinary  temperatures.  It  turns  lemon  yellow  at  118°C.  and  melts 
to  a  clear  dark  red  liquid  at  155°  C.  The  same  indicator  cannot  be 
used  twice  within  24  hours. 

The  salt  is  prepared  by  heating  in  a  dry  state  intimately 
mixed  mercuric  chloride  (2'7  gm.)  and  mercuric  iodide  (4-5  gm.)  in 
molecular  proportions  for  six  to  eight  hours  at  150°  to  160°  C.  At 
the  end  of  the  heating  the  product  should  be  powdered  and  kept  dry 
till  sealed  up  as  indicated.* 

Calculation  of  results     Example  : 
Urine  diluted  1  in  1 0. 
Height  of  standard  _  20mm. 
Height  of  unknown       18  mm. 

So  O'l  c.c.  urine  contain  I'll  mg.  N  as  urea  and  ammonia. 

*  These  indicators  can  be  obtained  from  Messrs.  Griffiths,  or  Messrs.  Baird 
and  Tatlock. 


CH.    X.]  UREA.  185 

So  100  c.c.  urine  contain  1000  X  1-11  mgm.  =  I'll  gm.  of  urea 
and  ammonia-N. 

Ammonia-N  was  found  to  be  045  gm.  per  100  c.c. 
Sourea-N  per  100  c.c.  =  Ml-0'045  =  1-065  gm. 

Urea  =  1-065  X  CO(^HA=  1-065  X  g  =  2'2S  per  cent. 

NOTES. — 1.  The  method  has  to  be  modified  for  diabetic  urine,  owing  to  the 
tendency  to  the  formation  with  the  sugar  of  stable  ureides  that  resist  decom- 
position. 

The  urine  is  diluted  100  times  and  1  c.c.  of  the  diluted  urine  decomposed  as 
above. 

The  ammonia  is  driven  into  another  tube  containing  about  2  c.c.  of  water 

N 

and  0'5c.c.  of  —  HC1.     To  this  tube  are  added  first  2  c.c.  of  water,  and  then 
10 

3  c.c.  of  1  in  5  Nessler's  solution.  The  coloured  solution  is  washed  into  a  10  c.c. 
measuring  flask  and  the  volume  made  up  to  10  c.c.  The  colour  is  determined 
against  that  of  the  usual  standard  containing  1  mgm.  of  nitrogen  per  100  c.c. 
of  solution. 

313.     The  estimation  of  urea  by  the  hypobromite  method. 

Remarks.  This  is  the  standard  method  for  the  clinical 
estimation  of  urea.  It  is  of  the  utmost  importance  for  the  student 
to  realise  that  the  method  is  essentially  inaccurate  and  may  lead  to 
very  erroneous  conclusions.  The  nitrogen  evolved  comes  from 
urea,  ammonia,  and  to  a  small  and  undetermined  extent  from 
creatinine  and  other  nitrogenous  constituents.  Further,  urea  does 
not  evolve  the  whole  of  its  nitrogen  in  the  form  of  gas,  so 
that  allowances  have  to  be  made.  Since  the  proportion  evolved 
varies  with  differences  in  the  composition  of  the  fluid  it  is  obvious 
that  no  certain  deductions  can  follow  such  a  determination.  It  is 
with  the  utmost  diffidence  that  the  method  is  given.  It  is  most 
certainly  not  to  be  recommended. 

Principle.  Urine  is  treated  with  an  alkaline  solution  of 
sodium  hypobromite  and  the  amount  of  urea  calculated  from  the 
volume  of  nitrogen  evolved. 

The  reaction  that  takes  place  is  as  follows  : — 
CO(NH2)2+3NaBrO  +  2  NaOH  =  N2  +  3  NaBr  +  NaaCO8+3  H2O. 


186 


ANALYSIS    OF    URINE. 


[CH.    X. 


Hence    60    grams   urea   evolve   28    grams    N.  =  2  X  11 '2    litres, 
and  1  gram  urea  evolves  373  c.c.  N. 

Practically  it  is  found  that  only  357  c.c.  are  evolved,  the  other 
4-4  per  cent,  of  the  nitrogen  being  converted  into  nitrates, 
cyanates,  etc. 

Apparatus.  See  fig.  10.  A  50  c.c.  burette  (a)  is  held  by  a 
clamp  in  a  tall  cylinder  of  water  (b).  The  upper  end  of  the  burette 
is  closed  by  a  tightly-fitting  rubber  stopper,  which  is  pierced  by  one 
limb  of  a  glass  T-piece.  The  upper  limb  of  the  T-piece  is  fitted 
with  a  short  length  of  pressure-tubing  carrying  a  screw-clamp  (e). 

The  side  limb  of  the  T-piece 
is  connected  by  about  two  feet 
of  small  rubber^  tubing  to  a 
glass  tube  piercing  the  well- 
fitting  rubber  stopper  of  a  wide- 
mouthed  bottle  (c)  of  about  60 
c.c.  capacity.  This  bottle  is 
placed  in  a  jar  of  water, 
supported  at  such  a  height  that 
the  burette  can  be  lifted  nearly 
out  of  the  tall  cylinder  without 
stretching  the  rubber  connec- 
tion. A  small  glass  bottle  or 
short  tube  of  10  to  15  c.c. 
capacity  is  also  required  (d). 
(For  the  method  of  preparing 
the  hypobromite  solution  see 
Ex.  249.) 


Fig.  10.    Apparatus  for  determination 
of  urea  by  hypobromite  method. 


Method  of  Analysis.  Place 
25  c.c.  of  freshly-prepared 
hypobromite  solution  in  (c). 
Put  5  c.c.  of  urine,  accurately  measured,  in  the  small  bottle  (j),and 
place  this  inside  the  other  by  means  of  a  pair  of  forceps,  taking 
great  care  not  to  upset  any  urine  into  the  hypobro  Fit  the 


CH.    X.J  UREA.  187 

rubber  cork  tightly  into  the  bottle  and  place  this  in  (6)  to  cool.  See 
that  the  burette  is  as  low  as  possible,  that  the  cylinder  has  sufficient 
water  in  it  to  reach  the  zero  graduation  of  the  burette,  and  that  the 
screw  clamp  is  open.  Leave  the  apparatus  for  about  a  minute  to 
cool  to  the  temperature  of  the  water ;  clamp  the  burette  in  such  a 
position  that  the  water  is  below  the  zero  mark,  and  then  screw  the 
clamp  on  the  rubber  tubing  as  tight  as  possible.  Note  down  on 
paper  the  level  of  the  water  in  the  tube,  keeping  the  eye  level  with 
the  meniscus.  Take  the  bottle  out  of  the  jar,  and  gently  tilt  it  so 
that  the  urine  flows  into  the  hypobromite. 

Gently  shake  the  bottle  from  side  to  side,  keeping  the  bottle 
upright  to  prevent  the  froth  from  being  forced  up  into  the  tube. 
Tilt  the  bottle  again  and  repeat  the  process  till  the  urine  and 
hypobromite  are  thoroughly  mixed.  Place  the  bottle  back  in  the 
jar  of  water  for  about  3  minutes  to  cool.  Raise  the  burette  till  the 
level  of  water  in  the  tube  is  the  same  as  that  outside,  the  gas  being 
thus  under  atmospheric  pressure.  Read  the  level  of  the  meniscus 
as  before  :  the  difference  in  the  two  readings  is  the  volume  of 
nitrogen  evolved.  Ascertain  the  temperature  of  the  water  and  the 
barometric  pressure. 

Calculation  of  results. 

Let  the  temperature  be  t°  C.,  the  tension  of  aqueous  vapour  at 
this  temperature  be  T  mm.  (See  Appendix),  and  the  barometric 
pressure  be  B  mm.  of  mercury.  Let  v  be  the  volume  of  nitrogen 
measured  under  the  conditions:  at  0°  C.  and  760  mm.  this  will 
become 

v  x  273  x  (B  -  T)  =    , 

(273  +  t)  x  760 
Now  357  c.c.  of  N  are  evolved  from  1  gram  of  urea. 

.*.  v'  c.c.  are  evolved  from  — —  gram  of  urea. 

v' 

-  •    5  c.c.  urine  contain  — —  gram  urea. 
357 

and  100  c.c.  urine  contain  — —  gram  urea. 


188  ANALYSIS    OF    URINE.  [cH.    X. 

NOTE. — Performing  these  two  calculations  in  one  operation  we  obtain  for 
the  percentage  of  urea 

v  X  (B  -  T    x  273  X  20  _  y  X  (B  -  T) 
(273  +  t)         760  X  357          (273  +  t) 

314.  The  estimation  of  uric  acid  by  the  Folin-Schaffer 
method. 

Principle.  The  mucoids  and  some  of  the  phosphates  are 
precipitated  by  ammonium  sulphate  containing  uranium  acetate  and 
acetic  acid.  The  filtrate  is  rendered  alkaline  by  ammonia. 

Ammonium  urate  separates  out.     This  is  washed  free  from  chlorides 

N 
with  ammonium  sulphate,  suspended  in  water  and  titrated  with  — 

potassium  permanganate. 

Preparation  of  Solutions. 

1.  Uranium  acetate  solution.     Dissolve  500  gm.  ammonium 
sulphate,  5  gm.  uranium  acetate  and  60  c.c.  of  10  per  cent,  acetic 
acid  in  650  c.c.  of  water.     The  volume  of  the  solution  is  almost 
exactly  1000  c.c. 

N 

2.  —  potassium  permanganate.     Dissolve   1*581   gm.  of  the 

pure  salt  in  distilled  water  and  make  the  volume  up  to  1000  c.c. 

Method.  200  c.c.  of  urine  are  treated  with  50  c.c.  of  the 
Folin-Schaffer  reagent,  allowed  to  stand  for  20  minutes  and  filtered 
through  a  dry  paper  into  a  dry  flask. 

Measure  125  c.c.  of  this  filtrate  into  a  beaker,  previously 
marked  at  the  100  c.c.  level  by  means  of  a  label,  add  5  c.c.  of 
concentrated  ammonia,  and  allow  it  to  stand  for  24  hours.  Care- 
fully filter  off  the  supernatant  fluid  through  a  hardened  filter  paper, 
and  wash  the  precipitate'  on  to  the  paper  with  10  per  cent, 
ammonium  sulphate.  Wash  the  precipitate  twice  more  with  this 
reagent  to  remove  the  chlorides. 

Remove  the  paper  from  the  funnel,  open  it,  and  by  a  fine  jet 
of  hot  water,  rinse  the  precipitate  back  into  the  beaker.  Cool 
under  the  tap  and  make  the  volume  up  to  100  c.c.  with  distilled 


CH.    X.]  URIC    ACID.  189 

water.     Add  15  c.c.  of  concentrated  sulphuric  acid  and  titrate  at 

N 
once,    without    cooling,    with  —   potassium  permanganate  from  a 

burette,  which  must  have  a  glass  tap. 

During  the  titration  the  fluid  in  the  flask  must  be  kept  in 
vigorous  movement.  Each  drop  of  the  permanganate  is  at  first 
discoloured  almost  immediately,  before  it  has  had  time  to  diffuse 
through  the  liquid  and  impart  to  it  a  pink  tinge.  The  first 
instantaneous  appearance  of  a  diffuse  flush  through  the  whole  body 
of  the  solution  marks  the  end  point  of  the  titration.  The  colour 
disappears  very  rapidly,  but  it  will  now  be  found  that  if  another 
drop  of  permanganate  be  added,  it  has  time  to  diffuse  through  the 
liquid,  before  it,  in  its  turn,  is  decolourised. 

Calculation  of  the  result. 

125  c.c.  of  the  fluid  taken  contains  100  c.c.  urine. 

N 
1  c.c  of  —  permanganate  =  0*00375  gm.  uric  acid. 

Add  to  the  result  0'003  gm.  for  the  100  c.c.  to  allow  for 
the  solubility  of  ammonium  urate  in  the  reagents. 

Example. 

24  hours  urine  =  1365  c.c. 
'124  c.c.  of  permanganate  required. 
Percentage  of  uric  acid  =  12*4  x  -00375  +  -003 
=  -0465  +  -003, 
=  -0495. 

Total  in  24  hours    =  '0495  X  13-65  =  '675  gm. 
Uric  acid-nitrogen  =  "675  X  £  =  -225  gm. 

315.     The    estimation    of    uric    acid    by    Folin's    micro- 
|  chemical  method. 

Principle.  Urine  is  evaporated ito  dryness  and  extracted  with 
ether  and  alcohol  to  remove  polyphenols.  The  residue  is  dissolved 
in  dilute  alkali  "and  treated  with  Folin's  uric  acid  reagent.  The 
fluid  becomes  coloured  blue  and  is  compared  colorimetrically  with 
a  standard  solution  of  uric  acid  similarly  treated. 


190 


ANALYSIS    OF    URINE.  [CH.    X. 


Preparation  of  Folins  reagent.     See  page  149. 

Method.  2  to  5  c.c.  of  urine  (depending  on  the  specific 
gravity)  are  measured  into  an  evaporating  basin,  a  single  drop  of  a 
saturated  solution  of  oxalic  acid  is  added  and  the  whole  evaporated  to 
complete  dryness  on  the  water  bath.  Allow  to  cool  and  add  10  c.c. 
of  a  mixture  of  2  parts  of  dry  ether  (distilled  over  sodium)  and 
1  part  of  pure  methyl  alcohol.*  Allow  to  stand  for  five  minutes 
without  stirring.  Carefully  pour  off  the  fluid  and  extract  similarly 
once  more.  To  the  residue  add  10  c.c.  of  water  and  a  drop  of 
saturated  sodium  carbonate  solution  and  stir  till  solution  is  complete. 
Add  2  c.c.  of  Folin's  reagent  and  then  20  c.c.  of  a  saturated  solution 
of  sodium  carbonate.  Transfer  the  blue  solution  to  a  100  c.c. 
graduated  flask,  wash  the  evaporating  basin  out  with,  water  into  the 
flask  and  make  the  volume  up  to  100  c.c.  By  means  of  Dubosq's 
colorimeter  (p.  191)  compare  the  colour  of  this  solution  with  that  of 
a  standard  solution  of  uric  acid  prepared  as  described  below. 

Preparation  of  the  standard  solution.  Weigh  out  250  mgms. 
of  Kahlbaum's  uric  acid.  Transfer  it  to  a  250  c.c.  measuring  flask 
by  means  of  25  to  50  c.c.  of  water.  Add  25  c.c.  of  a  04  per  cent, 
solution  of  lithium  carbonate  and  shake  at  intervals  for  an  hour 
before  making  the  volume  up  to  250  c.c.  The  solution  does  not 
keep  for  longer  than  5  or  6  days. 

1  c.c.  of  this  solution  is  carefully  measured  by  means  of  a 
reliable  pipette  into  a  100  c.c.  measuring  flask,  10  c.c.  of  water, 
2  c.c.  of  Folin's  reagent  and  20  c.c.  of  saturated  sodium  carbonate 
solution  are  added  and  the  volume  made  up  to  the  mark  with  water. 
The  reagent  and  sodium  carbonate  should  be  added  as  nearly 
simultaneously  as  practicable  both  to  the  unknown  and  to  the  standard 
solution.  Five  minutes  is  the  maximum  allowable  interval. 

Calculation  of  results. 

Set  the  standard  at  a  depth  of  x  mm. 

*  95  per  cent,   alcohol  can  be  used  as  a  substitute  for  the  ether-alcohol 
mixture,  but  it  involves  the  risk  of  a  slight  loss  of  uric  acid. 


CH.    X.] 


CREATININE. 


191 


Let  the  depth  of  the  unknown  be  y  mm.  when  an  equality  of 
tint  is  obtained. 

Suppose  3  c.c.  of  urine  were  taken. 

oc 
Then  3  c.c.  of  urine  contain  —  mgm.  uric  acid. 

y 


So  100  c.c.  contain 


mgm. 


316.     The  estimation  of  creatinine  by  Folin's  method. 

Principle.  Urine  is  treated  with  picric  acid  and  caustic  soda. 
The  creatinine  yields  a  colour  reaction  (see  Ex.  271),  the  intensity 
of  which  is  compared  with  that  of  a  standard  solution  of  potassium 
dichromate  by  means  of  a  colorimeter. 


Fig.   11. — Dubosq's  Colorimeter. 
Inset  shows  construction  of  vernier  scale. 


192 


ANALYSIS    OF    URINE. 


[CH.   X. 


0   *r 


Apparatus  required.      A  colorimeter.     Dubosq's  is  the  most 
convenient.     It  is  illustrated  in  Fig.  11.     The  solutions  to  be  com- 
pared are  placed  in  the  tubes  B.     The  tubes  D  are  sealed  at  the 
bottom  by  glass  plates  and  are  immersed 
in  the  solutions,  the  depths  of  which  are 
indicated    by   a  scale   and   vernier   at   the 
back   of    the    instrument.      Diffused   day- 
light is  reflected  through  the  solutions  by 
means  of  the  opal  glass    A,  the  path  of 
the   rays  being   shewn   in    Fig.    12.     The 
heights  of   the   tubes   D  are  so  adjusted 
that  an  exact  equality  of  tint  is  obtained. 

Before  using  the  colorimeter  it  is 
important  to  test  it  by  placing  the  bottom 
of  D  in  contact  with  the  bottom  of  B. 
Both  indicators  should  then  be  at  zero. 
Then  fill  the  tubes  B  with  the  standard 
dichromate  solution.  Place  one  of  the 
tubes  at  a  depth  of  10  mm.  Now  look 
through  O  and  adjust  the  level  of  the 
other  tube  until  the  two  tints  match.  The 
depth  of  this  solution  should  also  be 
10  mm.  Several  determinations  should 
be  made,  moving  the  second  tube  both 
from  above  and  below.  The  readings 
should  not  differ  by  more  than  0*3  mm. 

Preparation  of  the  dichromate 
solution.  Dissolve  24-55  gm.  of  pure 
potassium  dichromate  in  water  and  make 
the  volume  up  to  1000  c.c. 

Method  of  analysis.  Measure  10  c.c. 
of  the  urine  with  a  pipette  into  a  500  c.c. 
measuring  flask.  Add  15  c.c.  of  saturated 

picric  acid  in  water  (about  1'2  per  cent.)  and  5  c.c.  of  10  per  cent, 
caustic  soda.     Mix  and  allow  to  stand  for  5   minutes.     Fill  the 


r 

0 

-l_ 

u-p- 

n  i 

i 
j^         . 

B 

=J  L=^ 

:fi 

"v  : 

Fig.  12. — Diagram  of  path 
of  rays  in  Dubosq  s 
Colorimeter.  Below 
are  representations  of 
the  appearance  of  the 
field  under  different 
conditions,  that  on  the 
left  with  no  fluid  in  B, 
and  that  on  the  right 
when  the  tints  are 
matched. 


CH.    X.]  ACIDITY.  193 

flask  up  to  the  mark  with  distilled  water  and  mix  well.  Immedi- 
ately compare  with  the  standard  solution.  Place  the  dichromate 
in  one  of  the  cells  B  and  the  creatinine  in  the  other.  Place  the 
dichromate  tube  at  a  depth  of  8  mm.  and  determine  the  position  of 
the  other  that  gives  exactly  the  same  tint.  Take  several  readings 
working  from  above  and  below  as  before. 

Calculation  of  results. 

When  10  mg.  creatine  are  treated  in  this  way  the  colour  of  a 
layer  8'1  mm.  in  depth  is  the  same  as  that  of  a  layer  of  8  mm.  of 
the  standard  dichromate  solution. 

If  the  depth  of  the  layer  be  x  mm.,  then  the    creatinine    in 

10  X  8-1 
10  c.c.  01  urine  =  -  mg. 

NOTE. — If  the  reading  be  less  than  5  mm.,  the  urine  must  be  carefully 
diluted  and  another  determination  made  using  less  urine.  Should  it  be  greater 
than  13  mm.,  20  c.c.  must  be  taken  instead  of  10. 

317.  The  estimation  of  the  titration  acidity  by  Folin's 
method.  Place  25  c.c.  of  urine  in  a  200  c.c.  Erlenmeyer  flask, 
add  15  gm.  of  finely  powdered  neutral  potassium  oxalate,  2  drops 

of  1  per  cent,  phenolphthalein  and  shake  the  mixture  vigorously  for 

N 

2  to  3  minutes.     Titrate  with  —  sodium  hydroxide,  until  a  per- 
manent faint  pink  colour  is  produced. 

N 
Calculation.     Express  the  result  in  terms  of  —  soda.     Thus 

if  7  c.c.  of  soda  are  required  for  25  c.c.  of  urine,  the  acidity  of  the 

N 
same  is  equivalent  to  28  c.c.  of  —  sodium  hydroxide  per  100  c.c. 

NOTES.— 1.  The  method  only  gives  approximate  results  owing  to  the 
difficulty  in  determining  the  end-point.  Since  the  point  varies  with  the  amount 
of  indicator  used,  this  must  be  kept  constant.  Each  individual  worker  should 
always  choose  the  end-point  that  he  can  most  readily  determine,  and  always 
proceed  to  this  point.  In  this  way  reliable  information  oan  be  obtained 
concerning  the  relative  titration  acidity  of  various  samples  of  urine. 

2.  For  the  estimation  of  the  true  acidity  (the  concentration  of  the  hydrogen 
ions)  see  page  129. 

O 


194  ANALYSIS    OF    URINE.  [CH.    X. 

3.  The  potassium  oxalate  is  added   to    precipitate   calcium   salts,    which 
interfere  with  the  sharpness  of  the  end-point  of  the  titration. 

4.  The  urine  thus  neutralised  can  be  used  for  the  determination    of   the 
ammonia  of  the  urine  by  the  formaldehyde  method.     (See  p.  181.) 

318.     The  estimation  of  chlorides  by  Yolhard's  method. 

Principle.  The  chlorides  are  precipitated  from  urine  by  a 
known  excess  of  standard  solution  of  silver  nitrate  in  the  presence 

of  nitric  acid.  The   excess   of  silver   is   estimated  in   an  aliquot 

portion  of   the  filtrate   by  titration  with   a    solution  of   potassium 

sulphocyanide,  that  has  been  previously  standardised  against  the 

silver  solution,  a  ferric   salt  being  used  as  an   indicator   in   both 
titrations. 

Reagents  required. 

(i)  Standard  silver  nitrate  solution  prepared  by  dissolving 
29*063  grams  of  pure  fused  silver  nitrate  in  distilled  water 
and  filling  up  accurately  to  one  litre.  The  solution  should 
be  kept  in  the  dark. 

1  c.c.  corresponds  to  '01  gram  NaCl  (-00606  gram  Cl). 

(ii)     Solution  of  potassium   sulphocyanide   made    by    dissolving 
8  grams  of  the  salt  in  a  litre  of  distilled  water. 

(iii)     Pure  nitric  acid,  quite  free  from  chlorine, 
(iv)     A  concentrated  solution  of  iron  alum. 

Standardisation  of  the  Sulphocyanide.  In  a  beaker  place 
10  c.c.  of  the  silver  nitrate,  accurately  measured  :  add  5  c.c.  of  pure 
nitric  acid,  5  c.c.  of  iron  alum  and  80  c.c  of  distilled  water.  Titrate 
the  whole  with  the  sulphocyanide  from  a  burette  until  a  faint 
permanent  red  tinge  is  obtained.  Note  the  amount  required  for 
the  10  c.c.  of  silver  nitrate. 

Method  of  Analysis.  In  a  100  c.c.  cylinder  or  measuring 
flask  place  10  c.c.  of  urine,  accurately  measured  by  a  pipette, 
20  c.c  of  the  standard  silver  solution,  also  accurately  measured,  about 
4  c.c.  of  pure  nitric  acid,  and  5  c.c.  of  the  iron  alum.  Add  distilled 
water  till  the  100  c.c.  mark  is  just  reached,  and  mix  thoroughly  by 


CH.    X.]  CHLORIDES.  195 

pouring  into  a  beaker  and  stirring  well.  Filter  off  the  precipitated 
silver  chloride  through  a  dry  paper  into  a  dry  vessel.  Of  the 
filtrate  take  50  c.c.,  accurately  measured,  and  titrate  it  with  the 
potassium  sulphocyanide  solution  till  a  faint  permanent  red  tinge 
is  obtained. 

NOTE. — It  is  very  important  to  remember  to  add  the  nitric  acid.  It  renders 
the  silver  chloride  insoluble  and  prevents  the  precipitation  of  the  silver 
compounds  of  the  purine  bases  in  those  cases  in  which  the  urine  is  alkaline. 

Calculation  of  results. 

Standardisation  of  sulphocyanide  shows  that 

1  c.c.   KCNS  =  xc.c.  standard  silver. 
Now  50  c.c.  of  urinary  filtrate  =  S  c.c.  KCNS. 
/.    100  c.c.  „         „  „         =  2S  c.c.  KCNS 

2  S  c.c.  KCNS  =  2  S  X  x  c.c.  of  standard  silver. 
We  added  20  c.c.  standard  silver  to  10  c.c.  urine. 
So  (20  -  2  S  X  x)  c.c.  have  been  precipitated  by  chlorides  in 
10  c.c.  urine. 

1  c.c.  standard  silver  =  '01  gram  NaCl. 

So  10  c.c.  urine  contain  (20  -  2S  X  x)  x  -01  gram  NaCl. 

Thus  percentage  is  obtained. 

Example. 

19-6  c.c.  KCNS  =  10  c.c.  AgNO3. 

So  1  c.c.  KCNS  =  j^g  c.c.  AgNO3  -  x. 

50  c.c.  urinary  filtrate  required  11'6  c.c.  KCNS  =  S. 
100  c.c.        „  „  „         23-2  c.c.  =  2  S. 

23-2  c.c.  KCNS  =  2*\*6™  =  H*  c.c.  AgNO., 

So  20  -  11-8  =  8-2  c.c.  AgNO3  =  NaCl  in  10  c.c.  urine. 

NaCl  in  10  c.c.  is  8'2  X  -01  =  '082. 

NaCl  in  100  c.c.  is  0'82  gram. 

319.    The  estimation  of  phosphates. 

Principle.  Urine  is  heated  to  boiling  point,  and  titrated 
whilst  hot  with  a  standard  solution  of  uranium  acetate,  which  gives 
a  precipitate  of  (UO2)HPO4  with  phosphates  in  acetic  acid  solution. 


196  ANALYSIS    OF    URINE.  [CH.    X. 

Cochineal  tincture  is  used  to  indicate  by  a  change  in  colour  when 
the  uranium  is  in  excess. 

Reagents    required. 

(i)  A  solution  containing  100  grams  of   sodium   acetate   and 
100  c.c.  of  strong  acetic  acid  to  a  litre  of  distilled  water. 

(ii)  Cochineal  tincture,  prepared  by  extracting  the  insects  with 
30  per  cent,  alcohol  and  filtering  after  two  days. 

(iii)  A  standard  solution  of  sodium  phosphate.  Dissolve  twelve 
grams  of  pure  sodium  phosphate  in  a  litre  of  distilled  water.  Take 
50  c.c.  of  the  filtered  solution  and  evaporate  it  to  dryness  in  a 
weighed  dish  or  crucible  on  a  water  bath.  When  dry,  raise  the 
temperature  to  about  130°  C.,  and  leave  for  some  hours.  Allow  the 
dish  to  cool  in  a  desiccator  and  weigh.  Let  x  be  the  weight  of  the 
pyrophosphate  obtained.  Then  to  every  100  c.c.  of  the  remaining 

solution  add  from  a  burette  ^.^^  JG-C-  of  distilled  water.  A 
solution  is  thus  obtained  of  such  a  strength  that  50  c.c.  =  '1  gram 
P205. 

It  is  more  convenient,  but  not  quite  so  accurate,  to  prepare  the 
standard  solution  by  dissolving  3'85  grams  of  KH2PO4  in  water  and 
making  the  volume  up  to  1  litre. 

(iv)  Standard  solution  of  uranium  acetate.  Dissolve  by  the  aid 
of  heat  36  grams  of  uranium  acetate  in  a  litre  of  water.  Allow  the 
solution  to  cool  and  then  filter.  Standardise  the  solution  as  follows  : 
In  a  beaker  place  50  c.c.  of  the  phosphate  solution,  add  5  c.c.  of  the 
sodium  acetate  solution  and  a  few  drops  of  the  cochineal  tincture. 
Bring  the  solution  to  the  boiling  point,  remove  the  flame  and  titrate 
with  the  uranium  acetate  solution  from  a  burette  till  the  red  tinge 
just  changes  to  a  green,  heating  the  mixture  to  boiling  before  the 
last  few  drops  are  added.  Suppose  x  c.c.  of  the  uranium  are 
necessary:  then  to  every  100  c.c.  of  the  uranium  solution  add 
(20  -  x)  X  100  c^  of  distilled  water-  A  standard  solution  of 

x 
uranium  acetate  is  thus  obtained,  of  which  1  c.c.  =  "005  gram  P2O,. 


CH.    X.]  SULPHATES.  197 

Method  of  Analysis.  In  a  beaker  of  about  100  c.c.  capacity 
place  50  c.c.  urine,  add  5  c.c.  of  the  sodium  acetate  solution  and  a 
few  drops  of  the  cochineal  tincture.  Have  a  burette  ready 
containing  the  standardised  uranium  acetate  solution.  Heat  the 
urine  to  boiling  point,  remove  the  flame  and  run  in  the  uranium 
acetate  as  long  as  a  precipitate  is  formed.  Heat  the  mixture  again 
just  to  boiling  point,  and  cautiously  add  uranium  acetate,  drop  by 
drop,  till  the  red  colour  is  converted  to  a  green. 

Calculation  of  results. 

1  c.c.  of  the  uranium  acetate  =  '005  gram  P2O5. 
Thus  if  50  c.c.  of  urine  require  31  c.c.  uranium,  the  percentage 
of  P3O5  is  2  X  31  X  -005  =  -31  gram. 

320.  The  estimation  of  total  sulphates  by  Folin's  method. 
Place  25  c.c.  of  urine  in  a  250  c.c.  Erlenmeyer  flask,  add  20  c.c. 

of  hydrochloric  acid  (l  volume  of  concentrated  HC1  to  4  volumes  of 
water)  and  boil  gently  for  30  minutes,  covering  the  mouth  of  the  flask 
with  a  small  watch  glass.  Cool  the  flask  under  the  tap  and  dilute 
to  about  150  c.c.  with  water.  Add  10  c.c.  of  5  per  cent,  barium 
chloride  solution  slowly,  drop  by  drop,  to  the  cold  solution,  which 
must  not  be  stirred  or  shaken  during  the  addition,  nor  for  at  least 
one  hour  after.  Then  shake  well,  filter  through  a  weighed  Gooch 
crucible,  wash  with  250  c.c.  of  cold  water,  dry  in  an  air  bath,  or 
over  a  very  low  flame.  Ignite,  cool  and  weigh. 

Calculation.     Weight  of  BaSO4  X  1-373  =  SO3  per  cent. 

NOTES. — Instead  of  using  a  Gooch  crucible  a  washed  filter  paper  (Shleicher 
and  Schiill,  No.  589,  blue  ribbon)  may  be  used. 

After  washing  and  drying  the  ignition  may  be  carried  out  in  a  platinum  or 
porcelain  crucible,  previously  weighed.  After  ignition,  the  ash  should  be 
treated  with  a  drop  of  25  per  cent,  sulphuric  acid,  cautiously  dried  and  heated 
again . 

A  correction  must  be  made  for  the  weight  of  the  ash  of  the  paper. 

321.  The  estimation  of  inorganic  sulphates  by  Folin's 
method. 

Place  25  c.c.  of  urine  and  100  c.c.  of  water  in  a  250  c.c. 
Erlenmeyer  flask.  Acidify  with  10  c.c.  of  hydrochloric  acid  (l 
volume  of  concentrated  HC1  to  4  volumes  of  water).  Add  10  c.c. 


198  ANALYSIS    OF    URINE.  [CH.    X. 

of  5  per  cent,  barium  chloride,  drop  by  drop,  as   in  the  previous 
exercise,  and  proceed  as  there  directed. 

Calculation.     The  same  as  for  total  sulphates. 
Ethereal  Sulphates. 

This  can  be  found  by  the  difference  total  sulphates  less 
inorganic  sulphates. 

322.  The    estimation    of    total    sulphur    by    Benedict's 
method. 

Place  10  c.c.  of  urine  in  a  small  (7-8  c.c.)  porcelain  evaporat- 
ing dish  and  add  5  c.c.  of  Benedict's  sulphur  reagent.  Evaporate 
over  a  free  flame,  keeping  the  solution  just  below  the  boiling  point, 
to  prevent  loss  by  spattering.  When  dry,  raise  the  flame  slightly 
until  the  entire  residue  has  blackened.  Raise  the  flame  still  more 
and  heat  to  redness  for  ten  minutes  after  the  black  residue  (which 
first  fuses)  has  become  dry.  Allow  the  dish  to  cool.  ^Add  10  to  20 
c.c.  of  1  in  4  hydrochloric  acid,  and  heat  again  till  the  residue  has 
completely  dissolved  to  a  clear  solution.  Wash  the  contents 
quantitatively  into  an  Erlenmeyer  flask,  and  dilute  with  cold  water 
to  100  to  150  c.c.  Add  10  c.c.  of  10  per  cent,  barium  chloride, 
drop  by  drop,  and  allow  to  stand  for  about  an  hour.  Shake 
thoroughly  and  proceed  as  in  Ex.  320. 

323.  The  estimation  of  albumin  by  Esbach's  method. 


Fill  the  albuminometer  to  the  mark  U 
with  urine.  Add  Esbach's  reagent  (Ex.  16) 
to  the  mark  R.  Stopper  the  tube,  and  invert 
it  slowly  several  times  to  mix  the  fluids. 
Allow  the  tube  to  stand  upright  for  24  hours. 

Calculation.  The  graduations  on  the 
albuminometer  indicate  grams  of  albumin  per 
litre. 


Fig.   13.     Esbach's 
albuminometer. 


CH.    X.]  ALBUMIN.  199 

324.    The  estimation  of  albumin  by  Scherer's  method. 

Measure  50  c.c.  of  urine  into  a  beaker.  Place  it  on  a  water 
bath  and  raise  the  temperature  to  50°  C.  Add  1  per  cent,  acetic 
acid,  drop  by  drop,  to  obtain  a  complete  separation  of  the  protein 
(care  must  be  taken  to  avoid  an  excess).  Raise  the  temperature  to 
boiling,  and  keep  it  so  for  a  few  minutes.  Filter  the  urine  through 
a  small  paper  that  has  previously  been  washed,  dried  and  weighed. 
Wash  the  precipitate  in  turn  with  hot  water,  95  per  cent,  alcohol 
and  ether.  Dry  the  paper  and  precipitate  in  an  air  bath  at  110°C. 
till  the  weight  is  constant.  The  weight  of  protein  in  50  c.c.  is 
obtained  by  subtracting  the  weight  of  the  paper. 


CHAPTER    XI. 

DETECTION    OF     SUBSTANCES    OF 
PHYSIOLOGICAL    INTEREST. 

A.    Fluids. 

1.  Neutralise    a    considerable    portion,    and    evaporate    it    to 
dryness,    completing    the    process    on    a    water    bath    to    prevent 
charring.     This  evaporation  to  dryness  is  only  necessary  in  tests 
for  such  substances  as  urea  and  the  sugars.     If  these  be  known 
to    be    absent    it    can    be    omitted.     It    is    as  well   to    start    this 
evaporation  as  soon  as  possible,  as  it  takes  a  considerable  time. 
Neutralisation    is    necessary    to    obviate    any    chemical    changes 
produced  by  boiling  acids  or  alkalies. 

2.  Note  any  characteristic  smell  of  urine,  bile,  etc. 

3.  Note  the  colour  and  appearance  of  the  fluid :  opalescence 
suggests    starch,  glycogen,  or  certain    protein  solutions;    coloured 
fluids  suggest  bile,  blood  or  urine. 

4.  Note  the  reaction  to  litmus.     An  acid  reaction  excludes 
the  presence   of   mucin,   nucleoproteins,  caseinogen,  and  usually, 
earthy  phosphates. 

5.  If  acid  test  for  free  HC1  by  Gunsberg's  test.     (Ex.  187A.) 

6.  Sprinkle  some  flowers  of    sulphur    on    the    surface    of    a 
portion   of  the   fluid  in  a  test-tube.     If  the  particles  fall  through 
the    surface,    bile    salts   are    present.     (Ex.    224.)      Confirm    by 
Pettenkofer's  test.     (Ex.    223.) 

7.  If    the    fluid    be    brown     or     green,     apply    Cole's    test 
(Ex.  227)  for  bile  pigments. 


CH.    XI.]  FLUIDS. 


201 


8.  If  the  fluid  be  red  or  brown,  examine  for  blood-pigments 
or  derivatives  by  Table  F. 

9.  If  there  are  any  reasons  for  suspecting  the  presence  of 
ferments,  examine  by  Table  G.     If  none  of  the  colour  reactions 
for  proteins  are  obtained,  ferments  are  probably  absent. 

10.  Examine  for  proteins  by  Millon's  and  the  biuret  reactions. 
(Ex.    2    and    4.)        If    they    be    present,    proceed  '  as    directed   in 
Table  A,  B  or  C,  according  to  the  reaction  of  the  fluid. 

11.  If  proteins  are  absent,  proceed  to  Table  E. 

12.  Test    for    uric    acid  if    the    fluid  be  alkaline,  neutral   or 
only  faintly  acid.   Acidify  with  a  drop  or  two  of  strong  hydrochloric 
acid ;  uric  acid  may  separate  out  as  a  crystalline  powder.    Make 
another  portion  of    the  solution    alkaline   with  ammonia,  saturate 
with  NH4C1  and  apply  the  murexide  reaction    to  the    precipitate 
thus  obtained.    (Ex.  261.) 

13.  If  the  fluid    be    alkaline,  treat    a    little  with    a   solution 
of    calcium    chloride.       A    white    curdy    precipitate    indicates   the 
presence  of    soaps.     (Their  presence    should  be  confirmed  by  the 
methods  given  in  Ex.  128.) 


202 


DETECTION    OF    SUBSTANCES. 


[CH.   XI, 


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204  DETECTION     OF    SUBSTANCES.  [cH.    XI. 

Table  D. 

Examination  of  Filtrate  B  for  albumoses,  peptones  and  gelatin, 


Treat  a  portion  with  caustic  soda  and  a  drop  of  copper  sulphate  solution. 


No  biuret 
reaction. 


Proteins 
absent. 


Positive   biuret  reaction.     To  portions  of  filtrate  B  apply 
Millon's  and  glyoxylic  tests. 


Negative 
reactions. 


Positive  reactions.  Saturate  filtrate  B  with 
ammonium  sulphate  by  heating  with  excess  of 
solid.  Cool  under  tap  and  filter. 


Gelatin 

present. 


Precipitate.  Mostly  stick- 
ing to  tube.  Wash  with 
cold  saturated  ammonium 
sulphate.  Dissolve  in  a 
little  boiling  water  and  cool 
under  tap.  To  portions 
apply  biuret  test  (using  40 
per  cent.  NaOH)  and  the 
glyoxylic  test.  If  both  are 
positive — 


Albumoses. 


Filtrate. 
Treat     3    c.c. 
with  6  c.c.  of 
40,   per    cent. 
NaOH   and  a 
drop  of  copper 
sulphate. 
Pink       colour 
indicates 


Peptones. 


Table  E. 

Examination  of  a  solution  for  carbohydrates  and  urea. 

If  proteins  be  present  they  must  be  removed,  as  far  as 
possible,  by  neutralising,  boiling  and  filtering. 

In  any  case  the  solution  tested  must  be  neutral. 

(a)  To  a  small  portion  add  diluted  iodine  drop  by  drop,  until 
an  excess  has  been  added.  If  a  pure  blue  colour  be  obtained  at  any 
stage  of  the  addition  of  iodine,  starch  is  present.  If  a  purple  or 
brown  colour  be  produced  and  the  fluid  be  quite  clear,  erythro-dextrin 


CH.    XI.]  CARBOHYDRATES    AND     UREA. 


205 


is  present  and  glycogen  absent.     If  a  blue  colour  be  produced,  or  if 
the  fluid  be  opalescent,  proceed  as  follows  : 

To  a  portion  of  the  fluid,  prepared  as  directed  above,  add  an 
equal  bulk  of  saturated  (NH4)2SO4,  shake  vigorously,  and  filter 
through  a  dry  paper  after  about  ten  minutes. 


Precipitate. 
Scrape  off  the  paper, 
dissolve  in  a  little  hot 
water,  cool  and  add  a 
drop  of  iodine.  A  blue 
colour  shows  the  pres- 
ence of  starch. 


Filtrate.  To  a  small  portion  add  a  drop  or  two 
of  iodine .  If  a  reddish  or  purple  colour  be  produced , 
glycogen  or  dextrin  is  present.  If  the  fluid  be 
opalescent  after  warming,  glycogen  is  present. 
Saturate  the  remainder  with  (NH4)2SO4  and  filter. 


Precipitate. 
Neglect. 


Filtrate.  Add  a  drop  of  diluted 
iodine,  a  red-brown  colour  shows 
the  presence  of  erythro-dextrin. 


(b)  Apply  Benedict's  (Ex.  68)  or  Fehling's  test  (Ex.  67)  for 
reducing   sugars.      Note   that   the   tests   do   not   succeed   in   the 
presence  of  any  considerable  amount  of  ammonium  salts.    Also  that 
if    albumoses,    peptones    or    gelatin    are    present    they    should    be 
removed  by  alcoholic  extraction  as  described  in  Ex.  55. 

(c)  If  a  reduction  be  obtained,  apply  Barfoed's  test  (Ex.  69) 
to  distinguish  between  mono-  and  di-saccharides.     The  osazone  test 
(Ex.  73)  also  can  be  applied  if  necessary. 

(d)  Test  for  cane-sugar  by  Exs.  74-76. 

(e)  Examine  for  urea. 

Apply  hypobromite  test.  (Ex.  249.)  Effervescence  indicates 
urea  or  ammonium  salts.  If  obtained  boil  with  a  little  strong 
sodium  hydroxide  :  if  a  marked  smell  of  ammonia  be  not  obtained, 
ammonium  salts  are  absent  and  urea  is  probably  present.  In  any 
case  attempt  to  obtain  crystals  of  urea  nitrate  by  the  following 
method  : 

Evaporate  the  fluid  to  dryness  on  a  water  bath.  Add  alcohol, 
stir  till  boiling,  rub  up  thoroughly  and  filter.  Evaporate  the  alcohol 
to  dryness  on  a  water  bath.  Add  a  few  drops  of  water  to  the  residue, 
transfer  a  drop  of  the  solution  to  a  slide,  add  a  drop  of  strong  nitric 
acid  and  examine  for  crystals  of  urea  nitrate.  (Ex.  244.) 


206 


DETECTION     OF    SUBSTANCES. 

Table  F. 


[CH.    XI, 


Examine  the  solution  spectroscopically  :  gradually  dilute  the 
solution,  noting  the  spectrum  at  all  stages  of  dilution. 

Take  the  reaction  of  the  undiluted  fluid  to  litmus  paper,  wash- 
ing the  surplus  off  the  paper  with  a  stream  of  distilled  water,  if  you 
are  unable  to  note  the  reaction  directly. 

If  the  fluid  be  neutral  or  alkaline,  treat  it  with  Stokes'  fluid  or 
warm  it  with  ammonium  sulphide,  and  note  whether  the  spectrum 
is  altered  by  reduction.  This  should  be  done  after  various  dilutions 
of  the  original  solution. 


Fluid  red 


Acid — Acid  haematoporphyrin,  two  bands.      (Ex.  220.) 


Neutral 


Dilute  till  two 

bands  are  well 

seen  and  then 

reduce. 


Oxy haemoglobin,  the  two  bands 
merge  into  one  faint  band.  (Ex. 
207.) 

CO-haemoglobin,  the  two  bands 
are  unaltered.  (Ex.  209.) 


Alkaline 


Alkaline  haematoporphyrin,  four  bands,  converted 
into  acid  haematoporphyrin  by  strong  acids.  (Exs. 
220  and  221.) 

Haemochromogen,  two  bands  in  green,  one  much 
more  distinct  than  the  other,  unaffected  by  reduc- 
ing reagents.  (Ex.  219.) 


Fluid  brown 


/Acid — Acid  haematin,  band  in  red.     (Ex.  215.) 

Neutral.  Methaemoglobin,  band  in  red  :  gives  spectrum  of 
oxyhaemoglobin  and  then  of  reduced  haemoglobin 
if  reduced.  (Ex.  213.) 


Alkaline 


Alkaline  haematoporphyrin — four  bands.   (Ex.  221.) 

Alkaline  haematin,  faint  band  in  red,  converted  to 
haemochromogen  by  reducing  reagents.  (Exs.  21 7, 
219.) 


CH.    XI.]  FERMENTS. 


207 


Table  G. 

Examination  of  a  solution  for  ferments. 
Take  the  reaction  of  the  fluid  to  litmus. 
I.     Markedly  acid. 

Examine  for  pepsin.     (Ex.  185.) 

Neutralise    very    carefully    and    examine    for    rennin. 

(Ex.   143.) 
II.     Faintly  acid  or  neutral. 

Examine  for  ptyalin.      (Ex.  177.) 

Examine  for  pepsin.       (Ex.  185.) 

Examine  for  rennin.       (Ex.  143.) 

Examine  for  trypsin.     (Ex.  190.) 

Examine  for  lipase.       (Ex.  116.) 
III.     Distinctly  alkaline. 

Examine  for  ptyalin.     (Ex.  177.) 

Examine  for  trypsin.     (Ex.  190.) 

Examine  for  lipase.        (Ex.116.) 
Perform  control  experiments  in  all  cases.     (See  Ex.  118.) 


A  few  special  hints  on  the  examination  of  physiological  fluids. 

1.  It  is  impossible  to  obtain  a  heat  coagulum  of  albumin  or 
globulin  in  an  acid  or  alkaline  fluid.     The  reaction  must  be  neutral 
or  only  very  faintly  acid. 

2.  A  little  litmus  solution  in  the  fluid  does  no  harm,  and  often 
reminds  one  that  the  reaction  changes  after  boiling  (owing  to  the 
evolution  of  CO2). 

3.  In  testing  for  peptones,  after  removing  the  albumoses  by 
saturation  with  ammonium  sulphate,  the  biuret  test  succeeds  only  if 
at  least  two  volumes  of  40  per  cent,  soda  are  used.    The  test  will  not 
be  obtained  with  the  ordinary  5  per  cent.  soda. 

4.  Gelatin  reacts  very  much  like  the  albumoses,  except  that 
it  does  not  yield  the  glyoxylic  reaction. 


208  DETECTION     OF    SUBSTANCES.  [CH.    XL 

5.  It  is  impossible  to  obtain  Fehling's  or  Benedict's  test  for 
the  reducing  sugars  in  the  presence  of  ammonia  or  ammonium  salts. 

6.  The    sugars  reduce  only  in  an  alkaline  medium.     If  the 
fluid    under    examination    be    acid,   it   must   be  neutralised   before 
boiling  with  the  Fehling's  or  Benedict's  solution. 

7.  In  testing  for  cane  sugar  do  not  forget  that  starch  and  the 
dextrins  are  hydrolysed  to  dextrose  by  boiling  acids.     But  whereas 
cane  sugar  is  hydrolysed  very  easily,  starch,  etc.,  are  only  slowly 
acted  on. 

8.  Starch,  glycogen  and  the  erythro- dextrins  do  not  give  any 
colour  with  iodine  solutions,  if  the  reaction  of  the  fluid  be  alkaline. 
If  this  be  the  case,  make  the  reaction  acid  with  acetic  acid. 

9.  The    proteins    interfere   with    the    iodine    tests   for   these 
substances,  and  should  therefore  as  far  as  possible  be  removed 
before  testing  for  the  polysaccharides. 

10.  Fat  is  insoluble  in  water,  so  do  not  waste  time  in  testing 
an  ordinary  solution  for  fats. 

11.  The  only  reliable  test  for  urea  is  to  obtain  crystals  of  the 
nitrate  or  oxalate.     In  this  connection  it  must  be  remembered  that 
urea  is   soluble  in    alcohol,    and  can  thus  be   separated  from   the 
proteins    and    other    substances    likely    to    interfere    with    crystal 
formation. 

12.  Ammonium  chloride  is  a  very  valuable  reagent  in  testing 
for   uric  acid   or  urates.     The  only  other  physiological  substance 
precipitated  by  it  is  soap.. 

13.  Never    omit    "  control  "     tests    when    investigating    the 
ferment  action  of  a  solution. 

14.  Use  "  carmine  fibrin  "  in  testing  for  pepsin  ;  never  when 
testing  for  trypsin. 

15.  In  testing  solutions  for  pigments,  examine  spectroscopically 
in  various  dilutions.     Note  the  reaction  of  the  fluid ;  it  is  no  good 
looking  for  haemochromogen  in  a  markedly  acid  solution. 


CH.    XI.]  SOLIDS. 


B.     Solids. 


209 


1.  Examine  a  little  microscopically,  both  dry  and  with  the 
addition    of    a    drop    of    water.      Look  for  starch  grains,   crystals 
of  urea,  uric  acid,  urates,  leucine,  tyrosine,  cholesterin,  and  haemin 
scales. 

2.  Heat  a  small  amount  of  the  solid  in  a  dry  tube,  at  first 
gently  and  then  more  strongly. 

(a)  If   sublimation  take  place  and  an  odour  of  amylamme  be 
given  off,  leucine  is  present. 

(b)  If  sublimation  take  place  and  a  strong  smell  of  ammonia 
be  evolved,  urea  is  indicated. 

(c)  A  smell  of  phenol  and  nitro-benzol  indicates  tyrosine. 

(d)  A  smell  of  burning  feathers  indicates  proteins,  gelatin,  etc. 

(e)  A  smell  of  acrolein  indicates  fats. 

3.  Boil  some  of  the  solid  with  a  small  amount  of  water  in 
a   tube,   cool  under  the  tap  and    leave  the   test-tube  in  a  beaker 
of  cold  water  for  10  minutes.      If  gelatin  be  present,  the  solution 
will    set  to  a  jelly.     (Starch,  if  present,  may  form  a  thick  paste, 
which  may  be  confused  with  the  clean  jelly  given  by  gelatin.     If 
the  tube  be  subsequently  placed  in  boiling  water,  gelatin  becomes 
quite  limpid,  whilst  starch  remains  thick.) 

4.  If  the  solid  be  of  a  dark  brown  or  red  colour,  boil  a  portion 
with    dilute    alkali,    filter,    heat    the    filtrate   with   Stokes'   fluid  or 
(NH4)2S,    and    examine    for    the    spectrum    of   haemochromogen. 
(Ex.    219.)      If    this   be   obtained,    the   solid  contains  dried  blood 
or  haematin.     Confirm  by  obtaining  haemin  crystals.     (Ex.  222.) 

5.  Examine  by  the   method   indicated   in  the  Table  on  the 
next  page. 


210 


DETECTION     OF    SUBSTANCES. 


[CH.    XI 


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APPENDIX. 


1  grain  =  -0648  gram. 

1  ounce  =  437-5  grains  =  28-3595  grams. 

1  Ib.  =  16  oz.  =  7000  grains  =  453 -5925  grams. 

1  gram  =  15-432  grains. 

1  kilogram  =  1000  grams  =  2  Ib.  3^  oz.  (approximately). 

1  minim  =  -059  c.c. 

1  fluid  drachm  =  60  minims  =  3-55  c.c. 

1  fluid  ounce  =  8  fluid  drachms  =  28-4  c.c. 

1  pint  =  20  fluid  oz.  =567-9  c.c. 

1  c.c.  =  16-9  minims. 

1  litre  =  1000  c.c.  =35-2  fluid  oz. 

1  gallon  =  8  pints  =  4-548  litres. 

1  inch  =  2-54  cm. 

1  foot  =30-48  cm. 

1  yard  =  91 -44  cm. 

1  cm.  =  -39  in. 

1  metre  =39-37  in. 

Conversions. 

To  convert  grams  per  100  c.c.  into  grains  per  fluid  ounce,  multiply  by  4-375. 

To  convert  grams  into  ounces,  multiply  by  10  and  divide  by  284. 

To  convert  litres  into  pints,  multiply  by  88  and  divide  by  50. 

To  convert  kilos  into  pounds,  multiply  by  1000  and  divide  by  454. 

To  convert  degrees  Fahrenheit  into  degrees  Centigrade,  subtract  32,  multiply  the 

remainder  by  5,  and  divide  the  result  by  9. 
To  convert  Centigrade  into  Fahrenheit,  multiply  by  9,  divide  by  5,  and  add  32. 


212 


APPENDIX. 


TENSION    OF    AQUEOUS    VAPOUR 

in    millimetres    of    mercury    from    8°    to    25°  C. 


°C.        mm.       °C.       mm. 

"C. 

mm. 

8         8-0        14         11-9 

20 

17-4 

8-5        8-3    ,    14-5       12-3        20'5 

17-9 

9         8-6       15        12-7        21 

18-5 

9-5        8-9        15-5 

13-1        21-5 

19-1 

10         9-2 

16 

13-5        22 

19-6 

10-5        9-5 

16-5 

14 

22-5 

20-2 

11         9-8 

17 

14-4        23 

20-9 

11-5       10-1 

17-5 

14-9        23-5 

21-5 

12         10-5 

18 

15-3        24   *i 

22-2 

12-5       10-8 

18-5       15-8 

24-5 

22-9 

13        11-2 

19 

16-3        25 

23-5 

13-5       11-5 

19-5       16-8 

INTERNATIONAL    ATOMIC    WEIGHTS. 


Revised 
0  =  16. 

Approx. 

Revised 
0  =  16. 

Approx. 

Barium          Ba. 

137-4 

137 

'Manganese  Mn. 

55-04 

55 

Bromine         Br. 

79-97 

80 

Nitrogen          N. 

14-01 

14 

Calcium         Ca. 

40-01 

40 

Oxygen            O. 

16 

16 

Carbon            C. 

12 

12 

Phosphorus    P. 

31-2 

31 

Chlorine         Cl. 

35-46 

35-5 

Potassium       K. 

38-6 

•      39 

Copper          Cu. 

63-59 

63 

Silver             Ag. 

107-96 

108 

Hydrogen       H  . 

1-008 

1 

Sodium          Xa. 

23 

23 

Iodine                I 

126-8 

127 

Sulphur            S. 

32-07 

32 

Iron                  Fe 

56-02 

56 

Uranium         U. 

238-5 

239 

Magnesium  Mg. 

24-36 

24 

APPENDIX.  213 

Preparation  of  Normal  Solutions  of  Acids  and  Alkalies. 

A  normal  solution  of  a  substance  contains  in  one  litre  that  weight  in  grams 
which  corresponds  to  1  equivalent  in  grams  (1-008)  of  available  hydrogen  or 
its  equivalent. 

Thus  normal  hydrochloric  acid  contains  35'46  +  T008  =  36'468  gm.  of  HC1 
per  litre. 

Normal    sulphuric  acid  contains  2  U7  +  64  _  ^g.Q^  gm    jj2so4 

per  litre. 

Other  normal  solutions  are  : — 

Crystallised  oxalic  acid  (COOH)2.2H2O  =  63-03  gm.  per  litre. 
Anhydrous  sodium  carbonate  =53 

Sodium  hydroxide  =  40-01     ,, 

Ammonia  =  17-034 

The  most  reliable  starting  point  for  the  preparation  of  the  standard 
solutions  is  anhydrous  sodium  carbonate,  obtained  by  igniting  sodium 
bicarbonate. 

Powdered  sodium  bicarbonate  in  a  platinum  dish  is  placed  in  an  air  bath 
previously  heated  to  200Q  C.  The  temperature  is  raised  to  270Q— 280°,  but  not 
more  than  300°  C.  It  is  kept  at  this  temperature  for  an  hour,  and  cooled  by 
placing  the  dish  in  a  desiccator.*  Before  it  is  quite  cold  it  is  transferred  to 
warm,  dry,  stoppered  weighing  tubes,  which  are  allowed  to  cool  in  the 
desiccator. 

Have  ready  an  approximately  normal  solution  of  sulphuric  acid.  This  is 
obtained  as  follows: — Weigh  104  grams  of  pure  sulphuric  acid* into  a  counter- 
balanced beaker,  pour  the  acid  into  a  litre  flask  containing  about  500  c.c.  of 
distilled  water,  rinse  the  beaker  out  several  times  with  water,  adding  the  rinsings 
to  the  flask,  make  up  to  1000  c.c.,  mix  and  allow  to  stand  several  hours  to 
acquire  room  temperature.  Transfer  to  a  dry  Winchester  quart  bottle  and  add 
1  litre  of  water. 

Weigh  one  of  the  tubes  of  sodium  carbonate,  transfer  2  or  3  grams  to  a 
200  c.c.  beaker  of  Jena  glass,  and  weigh  the  tube  again  to  determine  the  exact 
amount  of  sodium  carbonate  taken.  Dissolve  this  in  80  to  100  c.c.  of  distilled 
water,  add  a  single  drop  of  methyl  orange  (0-1  per  cent.)  and  titrate  with  the 
sulphuric  acid  from  a  burette  till  the  mixture  just  turns  pink. 

Suppose  that  2-55  gm.  of  sodium  carbonate  are  neutralised  by  42-4  c.c.  of 
the  sulphuric  acid. 

Then  2'55  :  5-3  =  42-4  :  x        =  88-1  c.c. 

That  is  88-1  c.c.  of  the  acid  =  100  c.c.  Normal 

And  1000  c.c =1COOX100CC 

8o'l 
«  1135  c.c. 


214  APPENDIX. 

To  one  litre  of  the  sulphuric  acid  add  135  c.c.  of  distilled  water  and  mix. 
The  solution  thus  obtained  should  be  normal  sulphuric  acid.  It  should  be 
tested  against  sodium  carbonate  as  above  directed. 

Normal  sodium  hydroxide  is  prepared  by  dissolving  85  gm.  of  pure  NaOH 
in  water,  and  making  the  volume  up  to  2  litres.     When  quite  cold,  fill  a  burette 
with  the  solution  and  titrate  20  c.c.  of  normal   sulphuric  acid,   using   methyl 
orange  as  an  indicator.     Suppose  19-6  c.c.  of  the  NaOH  are  required, 
Then  19-6  c.c.  has  to  be  diluted  to  make  20  c.c. 

So   1    ,  —  c.c. 

19-6 

And  1000  c.c.  20  X  100°  c.c. 

19-6 

-  1020  c.c. 

To  1  litre  of  the  sodium  hydroxide  solution,  add  20  c.c.  of  distilled  water 
arid  mix.  Check  the  mixture  against  the  normal  sulphuric  acid. 

Thus  having  normal  solutions  of  an  acid  and  an  alkali,  normal  solutions  of 
other  acids  and  alkalies  can  readily  be  prepared. 

Strengths  of  Acids  and  Alkalies. 

ACIDS. 

100  c.c.  contain 

Acetic  acid,  glacial,  sp.  gr.  1-06       ...         ...         ...  ...  -     111-1  gm. 

Acetic  acid,  "strong"  ...          ...        "  ..          ...  ...  33  gm. 

Acetic  acid,  1  per  cent.          ...          ...          ...          ...  ...  1  gm. 

(9  c.c.  of  glacial  acetic  acid  made  up  to  1  litre) 

Hydrochloric  acid,  concentrated,  sp.  gr.  1-16       ...  ...  36-6  gm. 

Hydrochloric  acid,  0-4  per  cent ...  ...   «         0-4  gm. 

(11  c.c.  cone.  HC1  added  to  1  litre) 

Nitric  acid,  fuming,  sp.  gr.  1-40       ...          ...          ...  ...  about  65  gm. 

Nitric  acid,  concentrated,  sp.  gr.  1-42         ...          ...  ...  99  gm. 

Sulphuric  acid,  concentrated,  sp.  gr.  1-84  ...         ...  ...         175'9  gm. 

ALKALIES. 

Ammonia,  concentrated,  sp.  gr.  -880  ...          ...          ...  31  gm. 

Sodium  hydroxide,  sp.  gr.  1-34        ...          ...          ...          ...  40  gm. 

(410  gm.  of  98%  NaOH  or 

426  gm.  of  94%  NaOH  made  up  to  1  litre) 


Phenyl-glucosazone  (Ex.  73). 
Fine  yellow  needles  in  fan-shaped  aggregates,  sheaves  or  crosses. 


Phenyl-lactosazone  (Ex.  84). 
Ovoid  or  spherical  clusters  of  fine  yellow  needles. 


Plienyl-maltosazone  (Ex.  81). 

Broad  yellow  plates,  either  singly  or  arranged  in  spherical  clusters. 

215 


Potato  Starch  (Ex.  85). 

Ovoid  or  elliptical  grains,  with  concentric  markings  and  an 
eccentric  hilum. 


Wheat  Starch  (Ex.  151). 
Small  circular  grains  with  a  central  hilum. 


Tyrosine  (Ex.  191). 
Feathery  masses  and  sheaves  of  fine  white  needles. 


Leucine  (Ex.  191). 

Rounded  cone    or  balls  with  a  radiating  striation. 
216 


Oxy  haemoglobin  (dog)  (Ex.  203). 
Thin  rhombic  prisms. 


Haemin   (Teichmann's  Crystals,   Ex.  222). 
Brown  rhombic  prisms. 


ChoL-sterin  (Ex.  229). 

Rhombic  plates,  often  incomplete. 

217 


Urea  crystallised  from  acetone  (Ex.  245). 
Long  four-sided  prisms  or  fine  needles. 


Urea,  crystallised  from  alcohol  (Ex.  246). 

Irregular  branching  masses. 

218 


Urea  Oxalate  (Ex.  243). 
Long  thin  flat  crystals,  often  in  bundles.     Rhombic  prisms. 


Urea  Nitrate  (Ex.  244). 

Octahedral,  lozenge-shaped,  or  hexagonal  plates,  striated  or  imbricated. 

219 


Uric  Acid  (Exs.  258,  270,. 

(a)  Rhombic  plates 

(b)  Irregular  forms,   such  as  dumb  bells,    whet-stones,   butcher-trays,    stars 

and  crosses 

220 


Urinary  Sediments  (page  169). 

(a)  Urates  (spheres  with  projecting  spines) 

(b)  Calcium  oxalate  (envelopes  or  dumb-bells) 

(c)  Calcium  hydrogen  phosphate  (stellar  phosphate) 

(d)  Ammonium-magnesium    phosphate     (triple    phosphate),    prisms    (coffin 

lids)  or  feathery  stars 

(c)    Calcium  carbonate,  dumb-bells  or  spheres  with  radiating  structure 

221 


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MORE    RECENT    METHODS. 


325.     The  Estimation  of  Glucose  by  Bang's  Method  II. 

Principle.  A  known  volume  of  the  sugar  solution 
boiled  with  an  alkaline  solution  of  cupric  chloride, 
uprous  chloride  is  thus  formed.  The  amount  of  cuprous 
iloride  is  determined  by  titration  with  a  standard 
)lution  of  iodine.  Prom  the  volume  of  iodine  required 
le  amount  of  glucose  in  the  solution  can  be  calculated. 

Preparation  of  Solutions. 

1.  Stock  copper  solution.     ID  a  litre  flask  place  700  c.c.  of  boiled  out, 
•Id  distilled  water.   Warm  to  about  30^0.    Add  160  gm.  of  pure  powdered 
>tassium  bicarbonate.     When  dissolved,  add  66  gm.  of  pure  potassium 
iloride.     Cool  and  add  100  gm.  of  potassium  carbonate.     Then  100  c.c.  of 
4-4  per  cent,  solution  of  pure  crystalline  copper  sulphate.      Allow  to 
and  for  a  while  and  fill  up  to  the  mark  with  boiled  out  distilled  water. 
Lgorous  shaking  of  the  fluid  must  be  avoided.     The  solution  should  not 
}  used  for  24  hours. 

2.  Diluted    copper    solution.      300  c.c.  of  the  stock    solution    are 
luted  to  1  litre  with  a  cold  saturated  solution  of  potassium  chloride. 
void  excessive  shaking  and  allow  to  stand  for  some  hours  before  use. 

3.  N/10  iodine  solution.     12-685  gm.  of  pure  resublimed  iodine  are 
eighed  out  from  a  stoppered  tube  into  25  gm.  of  potassium  iodide  in 
'0  c.c.  of  distilled  water.     The  volume  is  made  up  to  1  litre  with  dis- 
Lled  water. 

4.  N/100  iodine  solution.      10  c.c.   of  N/10   iodine  are    diluted   to 
0  c.c.  with  boiled  out  distilled  water.     The  solution  is  only  stable  for 
:  hours. 

5.  Soluble  Starch.     1  gm.  of  Kahlbaum's  soluble  starch  is  dissolved 
100  c.c.  of  a  boiling  solution  of  potassium  chloride,  saturated  at  room 

mperature.    After  cooling  the  volume  is  made  up  to  100  c.c.    with 
stilled  water. 

Q 


226 


MORE     RECENT    METHODS. 


Method.  The  flange  of  the  neck  of  a  100  c.c.  Jena  flask  is 
cut  off  by  means  of  a  file.  The  neck  is  fitted  with  a  piece  of  rubber 
tubing  4  to  5  cm.  in  length  and  about  3  mm.  thick.  This  rubber 
collar  should  extend  about  2  cm.  above  the  neck.  (See  Fig.  14.) 

In  the  flask  place  O'l  to  2  c.c.  of  the  sugar  solution,  containing 
not  more  than  10  mg.  of  glucose.  Add  55  c.c.  of  the  diluted 
copper  solution.  Heat  to  boiling  on  a  piece 
of  asbestos  gauze  over  a  burner.  Allow  the 
boiling  to  continue  for  exactly  3  minutes. 
Seal  the  rubber  tubing  completely  by  means 
of  a  screw  clip,  remove  the  flask  from  the 
gauze  and  cool  it  by  immersion  in  cold 
water  for  about  1  minute.  Loosen  the  screw 
to  admit  air,  remove  the  rubber  .tubing,  add 
4  or  5  drops  of  the  soluble  starch  and  titrate 
at  once  with  the  N/100  iodine.  Vigorous 
shaking  must  be  carefully  avoided.  The 
addition  of  iodine  to  the  mixture  gives  rise  to 
a  deep  blue  which  is  at  first  rapidly  dis- 
charged. The  titration  is  completed  when  the 
"  starch-blue  "  tinge  is  permanent  for  20  sees. 

It  is  essential  that  the  titration  be  conducted  rapidly  to  minimise 
the  re-oxidation  of  cuprous  to  cupric  chloride  by  the  air.  It  is 
convenient,  but  not  absolutely  essential,  to  titrate  in  an  atmosphere 
of  CO2,  as  described  in  the  method  for  the  analysis  of  sugar  in 
blood  (p.  235). 

Calculation,     c.c.  of  iodine  divided  by  2'7  (or  multiplied  by  0'3704)  =  mg. 
of  glucose  in  the  volume  of  solution  taken. 

NOTES. — 1.     The  solution  must  not  contain  proteins. 

2.  The  method  is  well  adapted  to  the  estimation  of  glucose  in  urine. 

3.  N/25  or  N/10  iodine  can  be  used  for  the  titration,  but  a  microburette 
is  necessary.     If  N/25  is  used,  divide  by  0'7  ;  if  N/10,  divide  by  0'285. 

4.  It  is  advisable  to  use  the  standard  heating  apparatus  devised  by  the 
author.     It  is  illustrated  and  described  in  fig.  15  (p.  229).     The  heating  should 
be  such  that  the  mixture  begins  to  boil  in  1 J  mins. 


Fig.  14.     Flask  fitted 
for  sugar  estimation. 


GLUCOSE     BY     COPPER-IODIDE.  227 

326.  The  estimation  of  glucose  by  the  Method  of  Amos 
Peters. 

Principle.  A  known  amount  of  the  sugar  solution  is 
boiled  with  a  measured  amount  of  an  alkaline  solution 
of  copper  sulphate.  The  cuprous  oxide  is  filtered  off 
and  the  copper  in  the  filtrate  determined  hy  treatment 
with  potassium  iodide  and  titration  of  the  iodine  liberated 
by  means  of  a  solution  of  sodium  thiosulphate. 

Solutions  required. 

1.  Copper  sulphate.     69-278  gm.  of  the  purest  crystalline  salt  CuSO4, 
5H20,  is  dissolved  in  water  and  the  volume  made  up  to  1  litre.    It  is 
advisable    to    use    Kahlbaum's    copper    sulphate    "  with    certificate    of 
analysis,"  and  to  allow  for   the   very  small  amount  of  impurity  stated 
on  the  certificate. 

2.  Alkaline  tartrate.     346  gm.  of  Rochelle  salt  and  250  gm.  of  pure 
potassium  hydroxide  are  dissolved  in  water  and  the  volume  made  up 
to  1  litre. 

3.  N/5   Sodium  thiosulphate.     99-2  gm.   of  the  purest  thiosulphate 
.are  dissolved  in  boiled  out  distilled  water  and  the  volume  made  up 
to  1  litre  with  boiled  out  distilled  water.     It  should  be  prepared  at  least  a 
week  before  it  is  standardised. 

4.  Potassium  iodide.     Saturated  solution.     100  gm.  of  the  solid  are 
treated  with  70c.c.  of  hot  distilled  water  and  the  solution  allowed  to  cool. 

5.  Soluble  starch.     Shake  1  gm.  of  Kahlbaum's  soluble  starch  with 
about  10  c.c.   of  distilled  water  and  pour  the  suspension  into  90  c.c.  of 
boiling  water. 

Standardisation  of  the  thiosulphate.  Measure  20  c.c.  of 
the  copper  sulphate  into  a  200  c.c.  Erlenmeyer  flask.  Add  40  c.c. 
•of  distilled  water  and  20  c.c.  of  strong  (33  per  cent.)  acetic  acid. 
Insert  a  thermometer  and  cool  or  warm  to  20°  C.  Run  in  about 
6'5  c.c.  of  the  saturated  potassium  iodide,  the  thermometer  being 
withdrawn  and  its  stem  washed  with  this  solution.  The  iodine 
liberated  is  titrated  at  once  with  the  thiosulphate.  When  approach- 
ing the  end  point  add  about  1  c.c.  of  the  soluble  starch.  The  colour 
•changes  to  a  chocolate  brown  when  very  near  the  end  point. 
This  is  best  determined  by  the  "  spot  test  "  method.  Allow  a 
•drop  of  the  thiosulphate  to  fall  on  the  quiet  surface  of  the  liquid. 


228  MORE     RECENT     METHODS. 

If  the  end  point  has  not  been  reached,  a  very  perceptible  white 
area  is  seen  around  the  drop.  This  is  very  readily  distinguished 
from  the  diminution  of  the  slightly  yellowish  colour  of  the  sus- 
pended cuprous  iodide.  The  volume  of  the  drop  delivered  by  the 
burette  must  be  deducted  from  the  total  volume  added. 

The  copper  value  of  the  thiosulphate  is  calculated  as  shewn  in 
the  following  example  :— 

20  c.c.  of  the  copper  sulphate  =  352'93  mg.  Cu. 
This  required  27'6  c.c.  of  thiosulphate. 

352-93 

So  1  cc.  of  thiosulphate  =     nn        =  1278  mg.  Cu. 

4/'o 

The  heating  apparatus.  Use  the  apparatus  shewn,  in  fig.  15. 
In  a  200  c.c.  Erlenmeyer  flask  of  Jena  glass,  and  of  about  6  cm. 
basal  diameter,  place  60  c.c.  of  distilled  water.  The  flask  is  fitted 
with  a  2-hole  rubber  stopper  carrying  a  thermometer  so  graduated 
that  the  stem  above  34°  C.  is  visible  above  the  upper  edge  of  the 
stopper.  The  lower  end  of  the  thermometer  should  be  about  2  mm. 
from  the  bottom  of  the  flask. 

Turn  on  the  tap  B  to  its  full  extent  and  light  the  flame  of 
a  Bunsen  or  Meker  burner  which  is  placed  under  a  piece  of 
asbestos  gauze  carried  by  an  adjustable  ring  stand.  The  gauze 
should  be  from  4  to  6  cm.  above  the  top  of  the  burner.  Tighten 
the  screw  A  till  the  pressure  is  reduced  about  one-third.  Allow 
the  gauze  to  get  thoroughly  heated  and  then  place  the  flask  in  the 
centre  of  the  heated  gauze.  By  means  of  a  stop-watch  note  the 
time  for  the  temperature  to  rise  from  35°  to  95°.  If  the  time  is 
greater  or  less  than  120  sees,  loosen  or  tighten  the  screw  A  and 
repeat  the  experiment  with  another  60  c.c.  of  distilled  water  until 
the  temperature  of  the  water  rises  from  35°  to  95°  in  120  ±  2  sees. 
The  height  of  the  ring  and  the  thickness  of  the  asbestos  should  be 
such  that  the  pressure  is  well  under  the  minimum  supplied  to  the 


STANDARD     HEATING     APPARATUS. 


229 


laboratory  and  yet  sufficient  to  prevent  any  risk  of  the  flame 
striking  back.  Note  the  manometer  reading.  The  standard  heat- 
ing power  can  be  rapidly  obtained  for  further  experiments  by 
adjusting  the  screw  A  so  that  the  manometer  shews  the  requisite 
pressure. 


Fig.  15.  Apparatus  for  maintaining  a  standard  heating  power.  The 
manometer  tube  contains  a  dilute  solution  of  eosin  or  other  dye.  ft  also 
contains  a  globule  of  mercury  which  nearly  fills  the  bottom  of  the  tube.  This 
prevents  the  rapid  oscillations  of  pressure  due  apparently  to  the  explosions  of 
local  gas  engines. 

Filtering  Apparatus,  It  is  convenient  to  use  the  apparatus 
shown  in  Fig.  16.  A  is  a  Jena  flask  of  200  c.c.  capacity.  Tube  B 
is  an  ordinary  calcium  chloride  tube.  The  lower  end  should  reach 
at  least  3  cm.  below  the  lower  edge  of  the  stopper  to  prevent  loss 
by  splashing  during  filtration.  The  filtering  mat  is  made  of  glass 
wool,  asbestos  fibre,  powdered  pumice  and  asbestos  fibre  added  in 


230 


MORE     RECENT     METHODS. 


that  order.  The  mat  should  be  washed  with  nitric  acid  and  then 
thoroughly  washed  with  water.  After  a  test  the  cuprous  oxide  on 
the  mat  is  dissolved  in  nitric  acid  diluted  with  an  equal  volume  of 
water  and  then  thoroughly  washed. 

An  ordinary  Gooch  crucible  can  be  used  with  a  mat  prepared 
in  the  same  way.     The  arrangement  is  shewn  in  Fig.  20,  p.  239. 

Method  of  Analysis.  Into 
a  200  c.c.  Erlenmeyer  flask 
measure  20  c.c.  of  the  standard 
copper  sulphate,  20  c.c.  of  the 
aklaline  tartrate  and  20  c.c.  of 
the  sugar  solution  (which  must 
not  contain  more  than  180  mg. 
of  glucose).  Fit  the  two-holed 
rubber  stopper  firmly  into  the 
neck  of  the  flask,  adjust  the 
thermometer  so  that  its  lower 
end  is  2  mm.  from  the  bottom 
of  the  flask  and  place  on  the 
heated  gauze.  Note  the  time 
when  the  mercury  indicates  a 
temperature  of  95°  C.  Allow  the 
heating  to  continue  for  exactly 
20  sees,  beyond  this.  Remove  the 

flask  by  gripping  the  rubber  stopper  and  swill  it  for  a  second  or 
two  under  the  tap  or  in  a  bowl  of  water.  The  reduction  of  the 
temperature  practically  stops  the  reduction.  Filter  the  hot  fluid  at 
once,  using  the  stem  of  the  thermometer  as  a  stirring  rod.  Wash 
the  flask  twice  with  about  7  c.c.  of  distilled  water.  Cool  the  filtrate 
by  holding  the  flask  under  the  tap.  Add  exactly  4  c.c.  of  strong 
sulphuric  acid,  insert  a  thermometer  and  cool  to  20°.  Add  6-5  to 
7  c.c.  of  the  saturated  solution  of  potassium  iodide,  washing  the 
stem  of  the  thermometer  with  this  solution.  Titrate  at  once  with 
the  standardised  solution  of  sodium  thiosulphate  as  described  above, 
using  soluble  starch  as  an  indicator  when  near  the  end  point. 


Fig.  16.     Filtering  apparatus  for 
reduced  copper. 


LACTOSE     BY     COPPER-IODIDE.  231 

Calculation  of  results.  From  the  amount  of  thiosulphate 
required  the  amount  of  copper  in  the  filtrate  is  determined. 
Knowing  the  amount  taken  (352-9  mg.),  the  amount  reduced  by 
the  sugar  can  be  calculated.  The  amount  of  glucose  corresponding 
to  this  copper  can  be  determined  by  a  reference  to  the  curve  in 
Fig.  17. 

Example.  The  copper  in  the  filtrate  required  14-62  c.c.  of 
thiosulphate. 

1  c.c.  of  thiosulphate  =  12-86  mg.  Cu. 

So  copper  in  filtrate  =  14-62  X  12-86  =  188-1  mg.  Cu. 

So  copper  reduced  by  glucose  in  20  c.c.  =  352-9  -  188-2  =  164-7  mg. 

From  the  curve  this  is  seen  to  correspond  to  86-3  mg.  glucose. 

So  20  c.c.  contain  86-3  mg.  glucose. 

So  100  c.c.  contain  431-5  mg.  glucose.      =  0-431  per  cent. 

NOTE. — If  the  amount  of  reduced  copper  is  between  60  and  200  mg.,  the 
amount  of  glucose  corresponding  to  this  can  be  obtained  by  multiplying  by 
0-522, 

327.  The  estimation  of  lactose  by  the  copper-iodide 
method. 

The  method  is  exactly  similar  to  that  described  in 
the  previous  exercise.  The  author  is  responsible  for 
the  copper  values  for  lactose.  They  are  represented 
graphically  in  Pig.  18. 

It  must  he  noted  that  the  results  are  given  as 
anhydrous  lactose  and  not  as  the  crystalline  hydrate. 

In  the  case  of  lactose  as  much  as  250  mg.  may  be 
present  in  the  20  c.c.  taken. 

The  copper  values  above  25  mg.  Cu.  can  be  converted 
to  anyhrous  lactose  by  the  use  of  the  following  formula  : 
mg.  anhydrous  lactose  =  1-25  +  mg.  Cu.  x  0-778. 


232 


MORE     RECENT     METHODS. 


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MICRO-ANALYSIS     OF     SUGAR. 


233 


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0140     70     oBHHB  '          -    --  J--r::::::::±±:::::::::  :::: 
0                10               20               30               40               5 
100            110             120             130            140            1J 
200            210             220            230            240            2J 
300            310             320            330            340            3£ 

:::::::::::::::::::::::;  ::::::::::::::::!  5:l  :. 
0  60  70  80  90  100 
)0  160  170  180  190  200 
)0  260  270  280  290  300 
0  360  370  380  390  400 

mg.  Cu. 
Fig.  18.     Curve  showing  amount  of  copper  reduced  by  lactose  anhydride. 


328.  The  micro-analysis  of  sugar  in  blood  by  Bang's 
Method. 

Principle.  A  few  drops  of  blood  are  drawn  up  on  a 
weighed  piece  of  special  absorbing  paper.  The  paper  is 
again  rapidly  weighed  on  a  microbalance  or  torsion 
balance.  The  proteins  are  coagulated  by  the  addition 
of  a  boiling  acidified  solution  of  potassium  chloride. 


234  MORE      RECENT     METHODS. 

The  sugar  diffuses  out  into  the  solution  and  is  estimated 
by  a  modification  of  Bang's  II.  method. 

Solutions  required. 

1.  Stock  copper  solution  (see  p.  225). 

2.  Acid  solution  of  potassium  chloride  obtained  by  mixing  1360  c.c. 
of  a  cold   saturated   solution   of    pure  potassium   chloride,    640   c.c.    of 
distilled  water  and  1-5  c.c.  of  25  per  cent,  hydrochloric  acid. 

3.  Soluble  starch  (see  p.  225). 

.  4.  N/200  iodine.  Obtained  by  diluting  5  c.c.  of  N/10  iodine  to  100  c.c. 
with  boiled  out  distilled  water.  It  may  also  be  prepared  by  treating 
2  gm.  of  potassium  iodide  with  1  to  2  c.c.  of  2  per  cent,  potassium  iodate, 
adding  5  c.c.  of  N/10  hydrochloric  acid,  and  making  the  volume  up  to- 
100  c.c.  with  boiled  out  distilled  water.  The  solution  must  be  freshly 
prepared  each  day. 

Method  of  Analysis. 

1.     To  obtain  a  measured  amount  of  blood. 

Special  absorbing  papers  can  be  obtained  from  Grave  ot 
Stockholm,  or  of  Messrs.  J.  J.  Griffin  &  Sons,  Kings  way,  London. 

A  paper  is  held  in  a  small  spring  clip  and  the  clip  and  paper 
are  weighed  together  on  a  torsion  balance  or  other  form  of  micro- 
balance. 

The  hand  of  the  subject  is  washed  in  warm  water  and  dried. 
The  subject  is  instructed  to  swing  the  arm  backwards  and  forwards, 
keeping  the  hand  as  low  as  possible.  The  finger  is  pricked  with 
a  sterile  bayonet-pointed  probe  on  the  back  of  the  finger  about 
£  inch  above  the  nail.  A  piece  of  rubber  tubing  is  wound  tightly 
round  the  middle  joint  of  the  finger.  On  firmly  flexing  the  finger 
the  blood  usually  wells  up  in  sufficient  amount.  The  blood  is 
taken  up  on  the  paper,  until  the  paper  is  fairly  covered.  It  is 
undesirable  to  have  the  paper  fully  saturated  with  blood.  The 
paper  and  clip  are  immediately  weighed  on  the  torsion  balance,  and 
the  weight  of  blood  taken  is  thus  known.  A  convenient  amount  is 
about  120  mg. 


MICRO-ANALYSIS     OF     SUGAR.  235 

2.  Coagulation  of  the  proteins.     Place  the  paper  in  a  clean,. 
dry,  rather  wide  test-tube.     Measure  6|  c.c.  of  the  acid  solution  of 
potassium  chloride  into  another  test-tube.     Boil  and  rapidly  pour 
the  vigorously  boiling  solution  on  to  the  paper  in  the  other  tube. 
Allow  the  tube  to  stand  for  at  least  30  minutes.     Transfer  the  fluid 
to  a  50  c.c.  Jena  flask  with  a  straight  neck,  i.e.  with  the  flange  cut 
off.     Wash  the  paper  with  another  6i  c.c.  of  the  acid  potassium 
chloride,  and  add  this  to  the  fluid  in  the  flask. 

3.  Method  of  heating  the  solution.     To  the  flask  add  1  c.c. 
of  the  copper  solution,  and  fit  on  to  the  neck  of  the  flask  a  piece  of 
rubber  tubing  about  3  mm.  thick  and  about  5  cm.  in  length  (see 
Fig.  14,  p.  226).     For  heating  it  is  advisable  to  use  the  apparatus 
designed  by  the  author  and  shewn  in  Fig.  15,  p.  229.     The  pressure 
of  gas  or  the  height  of  the  asbestos  gauze  must  be  so  adjusted  that 
the    solution  begins  to   boil   in'  1|  minutes  +  5  sees.     Allow  the 
solution  to  boil  for  exactly  2  minutes.     Just  before  the  two  minutes 
is  completed,  commence  to  tighten  the  screw   clip  C.     When  the 
time  has  expired  tighten  the  clip  very  firmly,  remove  the  flask  at 
once  and  plunge  it  into  cold  water.     Allow  it  to  cool  in  a  stream  of 
water  for  about  1  minute. 

NOTE.— A  special  pair  of  forceps  has  been  devised  for  holding  the  flask 
and  sealing  the  rubber  tube. 

4.  Titration  of  the  fluid.     Owing  to  the  rapidity  with  which 
cuprous  chloride  is  oxidised  it  is  necessary  to  exclude  air  by  means 
of  an  atmosphere  of  CO2.     This  is  best  accomplished  by  means  of 
the  simple  apparatus  shewn  in  Fig.  19.     The  CO2  can  be  obtained 
from  a  cylinder  or  by  the  action  of  HC1  on  marble  in  a  Kipp's 
apparatus.     Loosen  the  clip,  remove  the  rubber,  and  immediately 
fit  in  the  tube  from  the  CO2  apparatus,  the  gas  having  previously 
been   turned  on.     Add    3   or  4  drops   of  the   starch   solution   and 
titrate    with    N/200    iodine    from   a    microburette.      The    titration 
is    completed    when    the    "starch-blue"    tint    persists    for   about 
20  seconds. 


236 


MORE     RECENT     METHODS. 


There  is  usually  no  difficulty  in  determining  the  difference 
between  the  greenish  blue  of  the  copper  and  the  bright  blue  of  the 
.starch-iodine  compound.  The  titration  should  be  done  against  a 
white  ground  and  preferably  by  daylight. 


Fig.  19.     Apparatus  for  titration  in  an  atmosphere  of  CO-2. 

A.  Wash  bottle  containing  water. 

B.  Tube  fitted  into  flask  so  that  the  iodine  can  fall  from  burette  directly 
into  the  fluid. 


5.     Calculation. 
c.c.  of  iodine  —  0-16  c.c. 


=  mg.  of  glucose  in  the  blood  taken. 


6.     Example. 

Weight  of  blood  =118  mg. 
Iodine  required  =  0-68  c.c. 
0-68-0-16      0-52 


=  0-13  mg.  glucose  in  118  mg.  blood. 
=  0*11  per  cent. 


MICRO-ANALYSIS     OF     CHLORIDES.  237 

NOTES. — 1.  The  special  papers,  micro-balance,  flasks,  rubber-tubing,  etc.r 
can  be  obtained  from  Grave,  of  Stockholm,  or  Messrs.  J.  J.  Griffin  and  Sons, 
Kings  way,  London.  The  latter  firm  will  also  supply  the  apparatus  shewn  in 
Figs.  14,  15,  16  and  19. 

2.  It  is  advisable  to  allow  the  blood  to  thoroughly  soak  into  the  paper 
before  coagulating  the  proteins.     But  a  delay  of  more  than  20  minutes  entails 
a  possibility  of  loss  of  sugar  by  glycolysis. 

3.  It  is  of  the  utmost  importance  to  use  pure  reagents,  clean  tubes,  flasks, 
etc.     Impurities  usually  result  in  the  figure  being  too  high. 

329.  The  micro-analysis  of  chlorides  in  blood  by  Bang's 
Method. 

Principle.  A  few  drops  of  blood  are  taken  up  from  a 
finger  prick  on  to  a  piece  of  weighed  absorbing  paper. 
The  paper  is  again  weighed  on  a  micro-balance  and  the 
weight  of  blood  thus  found.  The  proteins  are  coagulated 
by  pouring  on  a  boiling  acid  solution  of  magnesium 
sulphate.  After  cooling,  2  c.c.  of  standard  silver  nitrate 
are  added  and  the  silver  chloride  filtered  off,  a  little 
kieselgur  being  added  to  aid  filtration.  The  silver  nitrate 
in  the  filtrate  is  treated  with  2  c.c.  of  a  standard  solution 
of  potassium  iodide  and  potassium  iodate  and  a  few  drops 
of  starch  solution.  The  iodate  yields  free  iodine  owing  to 
the  presence  of  the  acid  in  the  coagulating  fluid.  The 
mixture  is  then  titrated  with  standard  silver  till  the  blue 
colour  disappears.  From  the  amount  of  silver  required 
to  effect  this,  the  amount  of  silver  in  the  filtrate  can  be 
determined.  Thus  the  amount  of  silver  that  has  dis- 
appeared in  the  formation  of  silver  chloride  can  be  calcu- 
lated, and  so  the  amount  of  chloride  in  the  blood  taken. 

Preparation  of  reagents. 

1.  N/100  silver  nitrate.     1-7  gm.  of  pure  silver  nitrate  are  dissolved 
in  distilled  water  and  the  volume  made  up  to  1  litre.    It  should  be  stored 
in  a  dark  bottle  and  kept  in  the  dark. 

2.  Iodide  and  iodate  solution.     0-015  gm.  of  potassium  iodate  and 
1-7  gm.  of  potassium  iodide  are  dissolved  in  water  and  the  volume  made 
up  to  1  litre.     2  c.c.  of  the  solution  are  measured  and  treated  with  10  c.c. 


238  MORE     RECENT     METHODS. 

of  solution  3  and  a  few  drops  of  the  soluble  starch.  This  mixture  is 
titrated  with  N/100  silver  nitrate  from  a  microburette  until  the  blue 
colour  disappears.  The  strength  of  the  solution  must  be  adjusted  by  the 
addition  of  water  or  of  a  dilute  solution  of  potassium  iodide  until  2  c.c. 
require  exactly  2  c.c.  of  the  silver  nitrate. 

3.  Acid  magnesium  sulphate.     2  litres  of  a  30  per  -cent,  solution  of 
magnesium  sulphate,  120  c.c.  of  strong  pure  nitric  acid  (sp.  gr.  1-42)  and 
280  c.c.  of  distilled  water  are  mixed. 

4.  Starch  solution.     1  gm.  of   Kahlbaum's   soluble   starch  are  sus- 
pended in  a  little  cold  water  and  poured  into  about  80  c.c.  of  boiling 
distilled  water.     20  gm.    of  pure  potassium  nitrate   are   added  to  the 
mixture.     The  solution  is  poured  into  a  number  of  small  sterile  bottles 
and  stoppered  whilst  still  hot.     In  this  way  the  solution  can  be  preserved 
for  a  considerable  time. 

5.  Kieselgur.      This   should  be   purified  by  heating  to  a  dull  red, 
washing  with  10  per  cent,   acetic  acid  and  then  with  distilled  water 
and  heating  again  to  redness. 

Measure  0-2  c.c.  of  distilled  water  into  a  tube  about  5  cm.  in  length 
and  about  5  mm .  bore  which  has  one  end  sealed.  Make  a*  jnark  with  a 
file  or  a  label  to  show  the  height  of  the  fluid.  Dry  the  tube  thoroughly. 
The  amount  of  kieselgur  for  each  experiment  is  measured  by  filling  the 
tube  to  the  mark  after  gently  tapping. 

Since  kieselgur  adsorbs  silver  nitrate  it  is  essential  to  determine  the 
amount  of  this  for  every  specimen  by  a  blank  experiment  conducted  as 
follows  :  To  10  c.c.  of  solution  3  add  2  c.c.  of  the  silver  nitrate  and  the 
measured  amount  of  kieselgur.  Shake  and  filter  through  a  Gooch  crucible 
as  described  below.  Wash  the  tube  and  the  kieselgur  twice  with  5  c.c.  of 
distilled  water.  To  the  filtrate  add  2  c.c.  of  the  solution  2  and  a  few  drops 
of  the  starch  solution.  Titrate  cautiously  with  N/100  silver  nitrate  from 
a  microburette  till  the  blue  colour  is  discharged.  The  amount  of  silver 
required  corresponds  to  the  amount  adsorbed  by  the  kieselgur. 

Method  of  Analysis. 

1.  To  obtain  a  measured  amount  of  blood.     The  procedure 
is  exactly  the  same  as  described  in  the  section  on  the  micro-analysis 
of  sugar  (p.  234). 

2.  Coagulation  of  the  proteins.     Place  the  paper  in  a  clean, 
dry,  rather  wide  test-tube.     Into  another  clean  tube  measure  10  c.c. 
of  the  acid  magnesium  sulphate  and  boil.     Whilst  vigorously  boiling 
pour  the  solution  on  to  the  paper  and  allow  it  to  stand  for  30 
minutes. 


MICRO-ANALYSIS     OF     CHLORIDES. 


239 


3.  Removal  of  the  silver  chloride.  To  the  tube,  still  con- 
taining the  paper,  add'  2  c.c.  of  N/100  silver  nitrate  and  the 
measured  amount  of  kieselgur.  Grease  the  rim  of  the  tube  with  a 
smear  of  vaseline  and  shake  vigorously.  Filter 
through  a  6  c.c.  Gooch  crucible  into  a  125  c.c. 
filtering  flask  connected  to  a  water  pump  (see 
Fig.  20).  The  bottom  of  the  crucible  is  covered 
with  a  piece  of  filter  paper  cut  a  trifle  larger  than 
the  bottom.  The  paper  is  then  washed  with  a 
little  water  which  is  sucked  through.  Care  must 
be  taken  to  see  that  all  the  perforations  of  the 
crucible  are  covered.  Empty  the  flask  and  before 
turning  on  the  pressure  fill  the  crucible  with  the 
mixture  in  the  tube,  being  careful  to  get  as  much 
kieselgur  as  possible  into  the  crucible.  Allow  a 
Fig.  20.  Gooch  few  drops  to  filter  through  before  turning  on  the 
crucible  and  pressure>  Filtration  is  rapid  and  the  filtrate  is 
filtering  apparatus  .  rr  -j.  •  1  j  •*  ,.  u 

for  micro-analysis     usually   quite   clear.       If  it    is   cloudy    it   must   be 

of  chlorides.  refiltered. 

4.  Washing  the  paper.     When  the  whole  of  the  fluid  has  been 
filtered  add  5  c.c.  of  distilled  water  to  the  tube,  shake  vigorously, 
pour  it  into  the  crucible   and  filter.     Repeat  this   operation  once 
more. 

5.  Titration    of  the  silver.     To  the   fluid   in  the  flask  add 
2  c.c.  of  the  iodide-iodate  solution  and  a  few  drops  of  the  starch 
solution.     Titrate  against  a  white  ground  with  N/100  silver  nitrate 
from  a  microburette.     The  blue  colour  gradually  disappears  as  the 
silver    iodide   is   formed  and   then    sharply    disappears,   leaving   a 
yellowish  green  solution. 

6.  Calculation  of  results.     2  c.c.  of  N/100  silver  =  2  c.c.  of 
the  iodide-iodate  solution.     The  volume  of  N/100  silver  required  to 
effect  the  disappearance  of  the  blue  colour  is  the  volume  of  silver 
that  has  disappeared  from  the  tube.     Of   this  volume   a   certain 


240  MORE     RECENT     METHODS. 

amount  has  been  adsorbed  by  the  kieselgur.  The  remainder  has 
formed  silver  chloride  with  the  chlorides  of  the  blood.  Since  1  c.c. 
of  N/100  silver  =  0'585  mg.  NaCl,  the  amount  of  NaCl  in  the 
blood  taken  can  be  calculated. 

7.     Example.     Weight  of  blood  taken  =  116  mg. 

Volume  of  N/100  si  ver  adsorbed  by  kieselgur 

-0-12  c.c. 
Volume  of  N/100  silver  required  for  titration 

=  1-21  c.c. 
Volume  of  N/100  silver  precipitated  as  silver 

chloride  =1-21  -0-12  =  1-09  c.c. 
NaCl  in  116  mg. blood- 1-09  X  0-585- -638  mg, 
NaCl  per  cent.  —  0'55. 

330.  The  estimation  of  chlorides  in  urine  hi  Larrson's 
Method. 

Principle.  The  pigments,  urates  and  other  inter- 
fering substances  are  removed  from  the  urine  by 
adsorption  with  blood  charcoal.  The  chlorides  are  esti- 
mated in  a  measured  amount  of  the  nitrate  by  direct 
titration  with  silver  nitrate,  using  potassium  chromate 
as  an  indicator. 

Reagents  required. 

1.  Standard  silver  nitrate  (see  p.  194J. 

2.  Merck's  pure  blood  charcoal  (Carbo  sanguinis  pnrissj.     Ordinary 
animal  charcoal  is  quite  useless. 

3.  A  5  per  cent,  solution  of  potassium  chromate. 

Method  of  analysis.  To  1  gm.  of  the  blood  charcoal  in  a 
dry  50  c.c.  flask  add  20  c.c.  of  the  urine.  Shake  vigorously  and 
repeat  the  shaking  at  intervals  for  10  minutes.  Filter  through  a 
small  dry  paper  into  a  dry  tube.  Measure  10  c.c.  of  the  filtrate  by 
means  of  a  pipette  and  transfer  it  to  a  small  beaker.  Add  5  or  6 
drops  of  the  chromate  and  titrate  with  the  standard  silver  nitrate 


ESTIMATION     OF     ACETONE.  241 

from  a  burette  until  the  end  point   is  reached,  as  indicated  by  the 
appearance  of  a  reddish -brown  colour. 

Calculation.     1  c.c.  of  silver  =  0-01  gm.  NaCl. 

Example.       10  c.c.  of  the  filtered  urine  required  10-6  c.c.  of  silver. 
So  10  c.c.  contain  10-6  x  0-01  gm.  NaCl. 
So  100  c.c.  contain  1-06  gm.  NaCl. 

331.  The  estimation  of  acetone  and  aceto- acetic  acid  in 
urine  by  the  method  of  Scott- Wilson. 

Principle.  The  urine  is  distilled  into  an  alkaline 
solution  of  silver  mercuric  cyanide.  The  aceto-acetic  acid 
is  decomposed  into  acetone,  which  passes  over  with  any 
preformed  acetone  into  the  cyanide.  An  insoluble  keto- 
mercuric-cyaiiide  compound  is  formed.  This  is  filtered 
off,  dissolved  in  acid,  and  the  amount  of  mercury 
determined  by  titratioii  with  standard  sulphocyanide. 
From  the  amount  of  mercury  present  the  total  amount 
of  acetone  in  the  urine  taken  can  be  calculated. 

Solutions  required. 

1.  Silver  mercury  cyanide.     Dissolve  9  gm.  of  pure  caustic  soda  and 
0-5  gm.  of  mercuric  cyanide  in  60  c.c.  of  distilled  water.     Add  20  c.c.  of 
0-7268  %  silver  nitrate  slowly  with  constant  stirring.     If  necessary  filter 
through   a  layer  of   washed  asbestos  in  a   Gooch  crucible.     The  silver 
nitrate  solution  is  prepared  by  diluting  5  c.c.  of  the  standard  silver  nitrate- 
used  for  Volhard's  method  (p.  194)  with  15  c.c.  of  distilled  water. 

2.  Acid  mixture.     Strong  nitric  acid          40  c.c. 

Strong  sulphuric  acid     5  c.c. 
Distilled  water  55  c.c. 

3.  N/5  potassium  permanganate.     6-324  gm.  of  permanganate  dis- 
solved in  water  and  the  volume  made  up  to  1  litre. 

4.  Standardised  solution  of  potassium  sulpho-cyanide.     Dilute  125 
c.c.  of  the  stock  solution  described  on  p.  194  to  make  1  litre  with  dis- 
tilled water.      Standardise  the  stock  solution   against  standard  silver 
nitrate   as  described  on  p.  194.      Divide  21'4  by  the  number  of  c.c.  of 
KCNS  required  for  10  c.c.  of  silver  nitrate. 

The  result  =  mg.  of  Hg  per  c.c.  of  the  diluted  KCNS. 

5.  A  saturated  aqueous  solution  of  iron  alum. 

R 


242  MORE     RECENT     METHODS. 

Method. 

1.  The  distillation  of  the  acetone.     Use  the  apparatus  shewn 
in  Fig.  21.     Into  flask  A  measure  an  amount  of  urine  that  yields 
between  O4  and  2  mg.  acetone.     Add  water  to  make  the  volume 
up  to  about  100  c.c.  and  then  1  c.c.  of  strong  sulphuric  acid.     Into 
flask  C  place  10  c.c.  of  40  per  cenr.  caustic  soda  and  a  few  glass 
beads.     Into  E  place  10  c.c.  or  more  of  the  silver  mercury  cyanide 
reagent  (there  must  be  at  least  25  c.c.  to  each  mg.  of   acetone). 
Close  the  tube  with  the  glass  rod  B  and  then  light  the  burners. 
The  soda  in  C  must  boil  before  the  fluid  in  A.     The  soda  is  kept 
just  boiling  whilst  A  is  allowed  to  boil  briskly.     The  first  appear- 
ance of  turbidity  in   E  is  noted  and  the    distillation    allowed   to 
proceed  for  another  six  minutes.      Remove  plug  B  and  turn  out 
the  flames.     Detach  tube  D  from  the  condenser  and  wa^sh  it  with 
a   jet   of   distilled   water   into   E.      Allow  the  fluid  to  stand  for 
10  minutes. 

2.  Filtration  of  the  mercury  compound.     Use  an  apparatus 
similar  to  that  shewn  in   Fig.   20,  p.  239.     The  Gooch  crucible 
should  be  of    10  c.c.  and  the  filtering  flask  of  250  c.c.  capacity. 
First  prepare  the  crucible.     Cut  a  filter  paper  slightly  larger  than 
the  bottom   of  the  crucible,  place  it  in  position  and  moisten   it. 
Then  pour  in  a  suspension  of  washed  asbestos  fibre  and  form  a 
mat  of  this  by  applying  suction.     A  small  amount  of  a  suspension 
of  washed  powdered  pumice  should  next  be  filtered  to  partly  close 
the  pores  of  the  filter. 

Filter  the  fluid  in  flask  E  through  this.  If  the  first  portions 
of  the  filtrate  are  cloudy  they  must  be  refiltered.  Wash  the 
precipitate  with  cold  water  till  free  from  silver. 

3.  Solution   of  the    mercury  compound.      By  means  of  a 
glass  rod  with  a  pointed  hook  transfer  the  filter  mat  to  an  Erlen- 
meyer  flask.     Place  the  crucible  in  the  neck  of  the  flask  and  wash 
it  through  into  the  flask  with   10  c.c.  of  the  acid  mixture.      The 
point  of  the  rod  should  also  be  washed  with  a  little  of  this  solution. 


ESTIMATION     OF    ACETONE.  243 

Add  1  c.c.  of  the  potassium  permanganate  and  boil  till  colourless. 
Add  more  of  the  permanganate,  a  few  drops  at  a  time,  till  the 
brown  tinge  persists  in  the  boiling  mixture  for  about  two  minutes. 
Discharge  the  colour  by  the  addition  of  a  few  drops  of  yellow 
nitric  acid. 


Fig.  21.     Apparatus  for  the  estimation  of  acetone. 

A.  Jena  flask.  B.  Solid  glass  rod  for  sealing  tube.  C.  Jena  flask. 
D.  Glass  tube  connected  by  rubber  to  condenser  tube.  E.  Erlenmeyer  flask. 
F.  Liebig  condenser. 

4.  Titration  of  the  mercury.  Cool  thoroughly  under  the 
tap  and  add  2  c.c.  of  the  iron  alum.  Titrate  with  the  diluted 
sulpho-cyanide  against  a  white  ground  until  a  very  faint  pinkish 
brown  tinge  is  permanent.  The  end  point  is  quite  sharp,  but  it 
must  be  noted  that  after  it  is  reached  a  considerable  amount  of 
sulpho-cyanide  can  be  added  without  appreciably  darkening  the 
tint. 

Calculation.  From  the  amount  of  sulpho-cyanide  required  the  amount  of 
mercury  can  be  found. 

1  mg.  Hg  —  '0606  mg.  acetone. 

Example.     10  c.c.  standard  silver  =  20'2  c.c.  of  stock  KCNS. 

21-4 
So  1  c.c.  dilute  KCNS  =  JJTZ  =  1'06  mg.  Hg. 


244  MORE     RECENT     METHODS. 

2  c.c.  of  diabetic  urine  taken.     Mercury  in  ppt.  required  24'2  c.c.  KCNS. 

So  mercury  =  24-2  x   1-06  =  25'65  mg. 

So  total  acetone  in  2  c.c.  =  25'65  x   -0606  =  1'55  mg. 

So  total  acetone  in  100  c.c.  =  1'55  x  50  =  77'5  mg. 

332.  The  microchemical  estimation  of  uric  acid  by  the 
Folin-Denis  Method. 

Principle.  The  uric  acid  is  precipitated  as  silver 
urate.  This  is  centrifuged  off,  decomposed  by  the  addition 
of  hydrogen  sulphide  and  treated  with  Folin's  uric  acid 
reagent.  The  solution  is  compared  colorimetrically  with 
a  stable  solution  of  uric  acid  in  formaldehyde. 

Solutions  required. 

1.  Silver  lactate.     A  3  per  cent,  solution  in  distilled  water. 

2.  Magnesia  mixture.     55  gm.  of   crystallised  magnesium  chloride 
and  70  gm.  of  ammonium  chloride  are  dissolved  in  650  c.c.  ot  water  and 
the  volume  made  up  to  1  litre  with  strong  ammonia. 

3.  Uric    acid-formaldehyde    solution.       One    gm.    of    uric    acid    is 
dissolved  in  a  graduated  litre  flask  by  means  of  200  c.c.  of  a  0-4  per  cent, 
solution  of  lithium  carbonate  (solution  is  effected  rather  slowly).     Add 
40  c.c.  of  40  per  cent,  formaldehyde  solution,  shake  and  allow  to  stand  for 
some  minutes.     Acidify  by  the  addition  of  20  c.c.  of  normal  acetic  acid  and 
make  up  to  1  litre  with  distilled  water.     The  solution  should  be  perfectly 
clear  and  must  stand  at  least  a  day  before  it  is  standardised. 

4.  Folin's  uric  acid  reagent.     See  page  149. 

5.  Sodium  carbonate.    A  saturated  solution. 

6.  A  freshly  prepared  saturated  solution  of  hydrogen  sulphide  in 
water. 

Standardisation  of  the  uric  acid — formaldehyde  solution. 

Prepare  a  fresh  standard  solution  of  uric  acid  as  described  on 
pape  190.  Measure  1  c.c.  of  this  by  means  of  a  standard  pipette 
into  a  50  c.c.  measuring  flask.  Add  2  c.c.  of  the  uric  acid  reagent, 
10  c.c.  of  a  saturated  solution  of  sodium  carbonate,  and  make  up  to 
the  mark  with  distilled  water.  As  nearly  simultaneously  as  possible 
treat  5  c.c.  of  the  uric  acid  formaldehyde  solution  in  another  50  c.c. 
flask  with  2  c.c.  of  the  reagent,  10  c.c.  of  saturated  sodium  carbonate 
and  water  up  to  the  mark.  Each  solution  is  well  mixed  and 
immediately  compared  in  a  Dubosq's  Colorimeter  (p.  192).  Place 


ESTIMATION     OF     URINARY     DIASTASE.  245 

the  tube  containing  the  standard  uric  acid  at  either  10  or  20  mm. 
and  determine  the  depth  of  the  other  tube  that  gives  exact  equality 
of  tint.  The  amount  of  reacting  uric  acid  in  the  uric  acid — 
formaldehyde  solution  can  then  be  calulated  as  shewn  in  the 
following  example  : 

Height  of  standard  solution  of  1  mg.  of  uric  acid  =  10  mm. 
Height  of  5c.c.  of  uric  acid-formaldehyde  solution  =10' 10  mm. 
So    5  c.c.    of    uric    acid-formaldehyde  corresponds   to 

10-10  =Q'99mg'  uric  acid- 

Or  if,  in  subsequent  estimations,  the  tube  containing  the  uric 
acid-formaldehyde  solution  be  kept  at  10*1  mm.,  this  corresponds 
to  1  mg.  of  uric  acid. 

Method  of  Analysis.  Into  the  tube  of  a  hand  centrifuge 
accurately  measure  1  to  2  c.c.  of  urine  and  add  water  to  make 
about  5  c.c.  Add  6  drops  of  the  silver  lactate,  2  drops  of  the 
magnesia  mixture  and  a  sufficient  amount  (10  to  20  drops)  of 
strong  ammonia  to  dissolve  the  silver  chloride.  Mix  and  centrifuge 
for  one  to  two  minutes.  Pour  off  the  supernatant  fluid  and  to  the 
residue  in  the  tube  add  six  drops  of  the  hydrogen  sulphide  solution 
and  one  drop  of  concentrated  hydrochloric  acid.  Place  the  tube  in 
a  beaker  of  boiling  water  for  seven  minutes  or  until  all  excess  of 
hydrogen  sulphide  has  been  removed.  Add  a  single  drop  of  a  0'5 
per  cent,  solution  of  lead  acetate.  If  a  brown  precipitate  is  pro- 
duced the  hydrogen  sulphide  has  not  been  removed,  and  the  tube 
must  be  replaced  in  the  boiling  water  bath. 

Cool  the  tube,  add  2  c.c.  of  the  uric  acid  reagent,  10  c.c.  of 
the  saturated  solution  of  sodium  carbonate,  transfer  to  a  50  c.c. 
graduated  flask  and  make  up  to  the  mark  with  distilled  water.  As 
nearly  simultaneously  as  possible  measure  5  c.c.  of  the  standardised 
uric  acid-formaldehyde  solution  into  another  50  c.c.  flask,  add  2  c.c. 
of  Folin's  reagent,  10  c.c.  of  saturated  sodium  carbonate  and  make 
up  to  the  mark  with  distilled  water.  Immediately  compare  the 


246  MORE     RECENT     METHODS. 

colour  of  the  two  solutions  by  means  of  a  Dubosq's  colorimeter. 
It  is  convenient  to  set  the  tube  containing  the  uric  acid-formal- 
dehyde solution  at  the  height  determined  when  it  was  standardised 
(10- 10  mm.  in  the  example  given  above). 

Calculation  and  Example. 

2  c.c.  of  urine  taken. 
Height  of  urine  tube  =  9'8  mm. 

Height  of  standard  tube  =  lO'l  mm.   (corresponds  to  10  mm.  of  a  solution 
of  1  mg.  uric  acid). 

So  uric  acid  in       2  c.c.  =  — —  mg.     =  1-02  mg. 

So  uric  acid  in  100  c.c.  =  1-02  x  50  =  51  mg. 

=  O'OSl  per  cent. 

333.  The  Estimation  of  the  diastatic  power  of  urine  by 
Wohlgemuth's  Method. 

Principle.  Varying  amounts  of  urine  are  treated  with 
a  given  amount  of  soluble  starch  for  30  minutes  at  38°  C. 
After  cooling,  a  drop  of  dilute  iodine  is  added  to  each  tube. 
The  tubes  that  contain  considerable  amounts  of  urine 
have  all  the  starch  digested  so  that  no  colour  is  obtained 
on  adding  the  iodine.  The  tube  with  the  smallest  amount 
of  urine  that  completely  digests  the  starch  is  found  and 
so  the  diastatic  value  calculated. 

Reagents  required. 

1.  Stock  solution  of  soluble  starch.    Accurately  weigh  out  2  gms.  of 
Kahlbaum's  soluble  starch  and  transfer  it  to  a  dry  test  tube.    Add  about 
10  c.c.  of  distilled  water  and  shake.     Pour  the  suspension  into  about  70  c.c. 
of  boiling  distilled  water  and  stir  well.     Wash  the  tube  three  successive 
times  with  5  c.c.  of  distilled  water,  transferring  the  washings  to  the 
boiling  solution.     Now  add  10  gms.  of  pure  sodium  chloride.    Allow  to 
cool  and  make  the  volume  up  to  100  c.c.  with  distilled  water.     The  solution 
is  stable  for  months. 

2.  One  per  mille  soluble  starch  in  0-5  per  cent,  sodium  chloride. 
5  c.c.  of  the  stock  are  diluted  with  distilled  water  to  make  100  c.c.     This 
solution  must  be  freshly  prepared  each  day. 

3.  N/50  iodine,  prepared  from  N/10  iodine  (see  page  225)  by  diluting 
2  c.c.  with  8  c.c.  of  distilled  water.     The  diluted  iodine  must  be  freshly 
prepared  each  day. 


ESTIMATION     OF     URINARY     DIASTASE. 


247 


Method.     Label  a  series  of  clean  dry  test-tubes  1  to  10. 

Into  the  tubes  measure  the  volume   of   urine  and  of  distilled 
water  stated  in  the  table. 


Tube. 

c.c.  of  Urine. 

c.c.  of 
Water. 

d^ 
30' 

Tube. 

c.c  of  Urine 
diluted  1  in  10 
with   water. 

c.c.  of 
Water. 

d™l 
30' 

1 

0-5 

0-5 

4 

6 

0-9 

0-1 

22-2 

2 

0-4 

0-6 

5 

7 

0-8 

0-2 

25 

3 

0-3 

07 

6-6 

8 

07 

0-3 

28-6 

4 

0-2 

0-8 

10 

9 

0-6 

0-4 

33-3 

5 

o-i 

0-9 

20 

10 

0-5 

0-5 

40 

To  each  tube  add  2  c.c.  of  the  one  per  mille  starch,  com- 
mencing with  tube  10.  Mix  the  contents  by  agitation  and  place  in 
a  thermostat  or  a  water  bath  at  38°  C.  for  exactly  30  minutes. 

Remove  the  tubes  and  place  them  in  a  beaker  of  cold  water  for 
3  minutes  to  cool. 

Arrange  the  tubes  in  order  in  a  stand. 

Commencing  with  tube  1  add  1  drop  of  the  N/50  iodine  to 
each  tube  and  carefully  note  the  colour  produced. 

Should  a  colour  be  produced  and  it  rapidly  fades,  add  1  more 
drop  of  iodine  to  each  tube. 

Note  the  tube  with  the  lowest  number  that  shows  a  blue  tinge. 
The  next  lower  tube  contains  an  amount  of  urine  that  completely 
digests  2  c.c.  of  O'l  per  cent,  starch  in  30  minutes  at  38°  C. 

From  this  the  number  of  c.c.  of  0'1%  starch  that  would  be 
digested  by  1  c.c.  of  urine  can  be  calculated.  This  index  is  written 

38° 
as  d30'. 

NOTES. — 1.  It  is  customary  to  use  a  freshly  prepared  0-1  per  cent, 
solution  of  starch  in  water  and  to  make  the  volume  of  the  urine  up  to  1  c.c. 
with  1  per  cent,  sodium  chloride.  The  author  has  determined  that  2  per  cent, 
starch  in  10  per  cent,  sodium  chloride  is  quite  stable  and  that  the  results 
obtained  agree  with  these  found  by  the  original  method.  It  is  suggested  that 
the  present  more  convenient  method  be  adopted  as  a  standard. 


248  MORE     RECENT     METHODS. 

38° 

2.  The  d  — -t  of  normal  urine  varies  between  5  and  20,  with  an  average 

of  10.     In  acute  pancreatitis  the  value  is  high  and  may  be  over  200.     In  such 
cases  the  urine  is  still  further  diluted  to  1  in  100  and  the  d  calculated. 

2 

Thus  if  0-006  c.c.  of  urine  is  required  d  =  ——  =333. 

*UUo 

3.  It  is  important  that  the  same  amount  of  iodine  be  added  to  each  tube. 
Very  uneven  results  are  obtained  if  varying  amounts  of  iodine  be  employed. 

4.  Samples  of  the  mixed  24  hours'  specimen. should  be  used. 

5.  The  diastase  in  the  urine  is  quite  stable  if  the  urine  be  preserved  by 
the  addition  of  toluol.     3  c.c.  are  ample  for  an  estimation. 

6.  The   pipettes   for   measuring  the   solutions   must   be   accurate    1    c.c. 
pipettes  graduated  to  1/100  c.c. 

334.     The  estimation*  of  pepsin  by  Fuld's  Method. 

Principle.  An  acid  solution  of  edestin  (the.  protein 
of  hemp  seeds)  is  precipitated  by  sodium  chloride:  the 
peptic  digestion  products  are  not  precipitated. 

Solutions  required. 

1.  Hydrochloric  acid.     Dilute  30  c.c.  of  N/10  HC1  to  100  c.c.  with 
distilled  water. 

2.  0-1  per  cent,  solution  of  edestin.     Dissolve  0-1  gm.   of  Merck's 
edestin  in  100  c.c.  of  the  hydrochloric  acid  at  boiling  point.     Cool  and 
make  up  to  100  c.c.  with  the  hydrochloric  acid.     If  the  solution  is  not 
clean  it  must  be  filtered. 

3.  Saturated  (33  per  cent.)  solution  of  sodium  chloride. 

Method.  Number  a  series  of  clean  tubes  from  1  to  10. 
Into  tubes  2  to  10  measure  1  c.c.  of  the  hydrochloric  acid.  Into 
tubes  1  and  2  measure  1  c.c.  of  the  gastric  juice.  Mix  the  fluid  in 
tube  2  and  transfer  1  c.c.  to  tube  3.  Mix  this  and  transfer  1  c.c. 
of  the  mixture  to  tube  4.  Proceed  in  this  way  till  each  tube 
contains  1  c.c.  of  fluid  and  each  tube  contains  one  half  of  the 
amount  of  enzyme  present  in  the  tube  with  the  next  lower  number. 
To  each  tube  add  1  c.c.  of  the  edestin  solution.  •  Mix  and  allow  the 
tubes  to  stand  at  room  temperature  (15  to  17°  C.)  for  30  minutes. 
To  each  tube  add  10  drops  of  the  sodium  chloride.  The  tubes 
with  low  numbers  are  probably  clear,  whilst  the  tubes  with  high 


COLE'S     TEST    FOR    GLUCOSE.  249 

numbers  are  cloudy.  Note  the  tube  with  the  lowest  number  that 
shews  a  cloud.  The  tube  with  the  number  next  below  it  has  an 
amount  of  gastric  juice  that  just  digests  2  c.c.  of  the  edestin  in  30 
minutes.  Thus  the  number  of  c.c.  of  edestin  digested  by  1  c.c.  of 
gastric  juice  can  be  calculated. 

16° 
This  is  best  denoted  by  pe  — ; 

Thus    if  tube   6   shews  a  cloud,   then   in  tube   5    (containing 
0-0625  c.c.   gastric  juice)   digestion    is    complete.     Supposing   the 

temperature  is  16°  C.  then  PC  ^  =  — |—  =  32. 

' 


335.    Cole's  test  for  small  amounts  of  glucose  in  urine. 

In  a  dry  boiling  tube  or  large  test-tube  place  about 
1  gm.  of  Merck's  pure  blood-charcoal.  Add  10  c.c.  of  the 
urine,  shake,  heat  to  boiling  and  then  cool  under  the  tap. 
Shake  at  intervals  for  5  minutes.  Filter  through  a  small 
paper  into  a  dry  test-tube.  To  the  nitrate  add  6  drops  of 
pure  glycerine  and  0-5  gm.  of  anhydrous  sodium  carbonate, 
Shake  and  heat  to  boiling.  Maintain  the  boiling  for 
exactly  50  sees.  Immediately  add  4  drops  of  a  5  per  cent, 
solution  of  crystalline  copper  sulphate,  shake  to  mix  and 
allow  the  tube  to  stand  without  further  heating  for  one 
minute.  With  normal  urine  the  fluid  remains  blue.  If 
glucose  is  present  to  the.  extent  of  0-03  per  cent,  above  the 
normal  amount  in  urine  the  blue  colour  is  discharged 
and  a  yellowish  precipitate  of  cuprous  hydroxide  forms. 

NOTES. — 1.  The  treatment  with  blood  charcoal  removes  practically  the 
whole  of  the  urates,  creatinine  and  pigments  that  interfere  with  Fehling's  test 
(see  small  print  on  p.  161).  It  also  adsorbs  so  much  of  the  normal  amount  of 
glucose  present  that  the  filtrate  from  normal  urine  fails  to  give  a  reduction. 

2.  Ordinary  animal  charcoal  is  quite  useless.     Merck's  "blood  charcoal, 
purified  by  acid  "  must  be  employed.     1  gm.  is  roughly  measured  by  piling  up 
about  half  the  large  blade  of  an  ordinary  pen-knife  twice  with  the  charcoal. 

3.  0-5  gm.  of  anhydrous  sodium  carbonate  is  carried  by  about  f  the  length 
of  a  large  blade  well  piled  up  once. 


250  MORE     RECENT     METHODS. 

4.  Should  the  specific  gravity  of  the  urine  exceed  1025  it  is  advisable  to 
use  5  c.c.  of  the  urine  +  5  c.c.  of  water. 

5.  The  test  is  not  given  by  chloroform  nor  by  glycuronates  :  it  is  given  by 
pentoses. 

6.  Should  there  be  any  reason  to  suspect  lactose  the  procedure  should  be 
modified  as  follows:    treat  20  c.c.   of  the  urine  with   1   gm.   of    charcoal  as 
described  above.     Treat  the  whole  of  the  filtrate  with  another  gm.  of  charcoal 
and  repeat  the  process.     To  5  c.c.  of  this  filtrate  add  the  glycerine  and  sodium 
carbonate  and  proceed  as  above  directed.     A  reduction  indicates  the  presence 
of  glucose,  the  whole  of  any  lactose  up  to  even  1  per  cent,  being  removed  by 
this  double  adsorption,  whilst  0-04  per  cent,  of  glucose  in  the  original  urine  still 
shows  in  the  filtrate. 

336.    On  the  detection  of  acetone  and  aceto-acetic  acid. 

It  is  now  recognised  that  Rothera's  test  (Ex.  300)  is  a 
test  for  aceto-acetic  acid  as  well  as  for  acetone.  It  is  also 
clear  that  fresh  pathological  urine  never  contains  acetone 
unless  aceto-acetic  acid  is  also  present. 

Rothera's  test  is  the  simplest  one  to  apply  to  urine 
for  the  recognition  of  the  presence  of  the  acetone  bodies. 
The  author  has  determined  that  it  is  not  necessary  to 
have  a  freshly  prepared  solution  of  nitroprusside.  A 
mixture  of  1000  parts  of  pure  solid  ammonium  sulphate 
and  one  part  of  solid  sodium  nitroprusside  is  prepared  by 
grinding  and  intimate  mixing.  The  test  can  then  be 
performed  as  follows :  in  a  test-tube  place  the  solid  for  a 
depth  of  2  inches.  In  another  similar  tube  place  a  like 
depth  of  the  urine.  Pour  the  urine  on  to  the  solid  and 
mix  by  repeatedly  pouring  from  tube  to  tube.  Add  2  c.c. 
of  strong  ammonia,  mix  and  allow  to  stand.  The  develop- 
ment of  a  permanganate  colour  indicates  the  presence  of 
acetone  bodies  in  the  urine. 

Hurtley  has  recently  described  the  following  test 
which  is  specific  for  aceto-acetic  acid. 

To  10  c.c.  of  urine  add  2-5  c.c.  of  concentrated  hydro- 
chloric acid  and  1  c.c.  of  a  freshly  prepared  1  per  cent, 
solution  of  sodium  nitrite.  Shake  and  allow  to  stand  for 


COLE'S     TEST     FOR     BILE     PIGMENTS.  251 

two  minutes.  Now  add  15  c.c.  of  strong  ammonia,  then 
5  c.c.  of  a  10  per  cent,  solution  of  ferrous  sulphate.  Shake, 
pour  into  a  large  hoiling  tube  and  allow  to  stand  undis- 
turbed. A  violet  or  purple  colour  slowly  develops  if 
aceto-acetic  acid  be  present.  The  speed  at  which  the 
colour  develops  depends  on  the  concentration  of  aceto 
acetic  acid.  With  small  amounts  the  colour  may  not 
develop  for  about  5  hours.  The  test  shows  in  a  dilution 
of  1  in  50,000. 

337.    Cole's  test  for  bile  pigments. 

The  following  method  has  recently  been  elaborated 
as  an  improvement  on  that  described  on  page  119. 

Boil  about  15  c.c.  of  the  fluid  or  suspected  urine  in 
a  test  tube.  Add  two  drops  of  a  saturated  solution  of 
magnesium  sulphate,  then  add  a  10  per  cent,  solution  of 
barium  chloride,  drop  by  drop,  boiling  between  each 
addition.  Continue  to  add  the  barium  chloride  until  no 
further  precipate  is  obtained.  Allow  the  tube  to  stand 
for  a  minute.  Pour  off  the  supernatant  fluid  as  cleanly 
as  possible  or  use  a  centrifuge.  To  the  precipitate 
add  3  to  5  c.c.  of  97  per  cent,  alcohol,  two  drops  of  strong 
sulphuric  acid,  and  two  drops  of  a  5  per  cent,  aqueous 
solution  of  potassium  chlorate.  Boil  for  half  a  minute 
and  allow  the  barium  sulphate  to  settle.  The  presence  of 
bile  pigments  is  indicated  by  the  alcoholic  solution  being 
coloured  a  greenish  blue. 

To  render  the  test  more  delicate,  pour  off  the  alcoholic 
solution  from  the  barium  sulphate  into  a  dry  tube.  Add 
about  one-third  its  volume  of  chloroform  and  mix.  To 
the  solution  add  about  an  equal  volume  of  water,  place 
the  thumb  on  the  tube,  invert  once  or  twice  and  allow 
the  chloroform  to  separate.  It  contains  the  bluish 
pigment  in  solution. 


252  MORE     RECENT     METHODS. 

338.     Preparation  of  haemin  crystals  (Nippe's  Method)- 

A  small  drop  of  blood  is  spread  to  form  a  film  on  a 
glass  slide  and  sloivly  evaporated  till  it  is  quite  dry.  To 
the  film  add  two  drops  of  a  solution  of  0-1  gm.  of  potassium 
chloride  in  glacial  acetic  acid.  Cover  with  a  slip  and  heat 
over  a  very  small  name  till  bubbles  appear  and  the 
solution  is  boiling.  Allow  a  drop  or  two  more  of  the 
reagent  to  run  under  the  cover  slip  and  examine  under  a 
microscope. 

NOTE. — The  advantage  of  the  method  is  that  it  is  rapid  and  that  crystals 
of  the  inorganic  chloride  do  not  separate.  It  is  very  important  not  to  burn  the 
blood  whilst  drying,  and  also  to  be  sure  that  the  solution  is  heated  to  boiling 
with  the  acid  mixture. 


Bial's  reagent  consists  of  1  to  1-5  gm.  orcine,  500  c.c.  of 
concentrated  hydrochloric  acid,  and  30  drops  of  a  1  per 
cent,  solution  of  ferric  chloride. 


Benedict's  sulphur  reagent  is : 

Crystallised  copper  nitrate,  200  gm. 
Potassium  chlorate,  50  gm. 
Distilled  water  to  1  litre. 


INDEX. 


Absorption  spectra,  108 

chart  of,  222 
Aceto-acetic  acid,  165,  250 

estimation  of,  241 
Acetone,  165,  250 

estimation  of,  241 
Achromic  point,  86 
Achroo-dextrin,  46,  87 
Acidity,  129 

estimation  of,  129,  193 

of  gastric  juice,  91 

of  urine,  129,  193 
Acid  haematin,  112 
Acid  haematoporphyrin,  113 
Acidosis,  153,  165 
Acid  phosphates,  38 
Acids,  standard,  213 
Acrolein,  64 
Adenine,  20 
Adler's  test,  159 
Alanine,  95 
Albumin 

boiling  test  for,  155 

Crystallisation  of,  16 

detection  of,  202 

heat  coagulation  of,  6 

in  urine,  155 

properties  of,  12,  14 

removal  of,  15 

serum-,  14 

tests  for,  12,  14,  202 
Albuminuria.  155 
Albumoses,  23 

detection  of,  204 

deutero-,  24 

formation  of,  24 

hetero-,  24 

in  urine,  156 

primary,  24 

proto-,  24 

secondary,  25 

separation  of,  24 
Alkalies,  standard,  213 
Alkaline  haematin,  112 
Alkajine  haematoporphyrin,  113 
Alkaloidal  reagents,  10 
Allantoin,  145 
Alloxan,  145,  149 


Alloxantin,  149 
Alpha-napthol  test,  40 
Amino-acids,  96 
Ammonia  in  urine,  153 

estimation  of,  179-181 
Ammonium  sulphate 

standard  solution  of,  178 
Amylase,  84 
Amylodextrin,  46 
Analysis  of  fluids,  199 

of  solids,  209 
Anti-ferments,  84,  100 
Aqueous  vapour,  tension  of,  212: 
Arginine,  96 
Atomic  weights,  21 

Bang's  method  for 

chlorides,  237 

glucose,  225 

glucose  in  blood,  233 
Barfoed's  test,  37 
Beckmann's  method,  126 
Bence-Jones'  protein,  157 
Benedict's  method  for  sugar,  52 

for  sulphur,  198 

for  urea,  181 

Benedict's  sulphur  reagent,  252 
Benedict's  test,  36,  161 
Bial's  test,  164 
Bial's  reagent,  252 
Bile,  115 
Bile  pigments,  118,  251 

in  urine,  159,  251 
Bile  salts,  114 

in  urine,  160 
Bilirubin,  118 

Biuret,  formation  of,  140,  143 
Biuret  reaction  for  proteins,  5 
Blood 

coagulation  of,  99 

haemolysis  of,  103 

in  urine,  158 

laking  of,  103 

pigments,  106,  108,  206 

plasma,  101,  102 

serum,  6 

stains,  detection  of,  114 
Bread,  73 


254 


INDEX. 


Bromine  reaction  for  tryptopbane,  97 
Briicke's  reagent,  11 


Calcium  phosphate 

in  milk,  69 

in  urine,  136 

in  urinary  sediments,  170 
Calcium  salts 

in  clotting  of  blood,  99 

in  clotting  of  milk,  70 

in  heat  coagulation  of  proteins,  9 

in  milk,  69 

in  urine,  133,  138 
Cane  sugar,  39 

estimation  of,  58 
Carbohydrates,  31 

detection  of,  204 

in  proteins,  6,  17 
Carbonic  oxide  haemoglobin,  110 
Carboxy-haemoglobin,  110 
Carmine  fibrin,  89 
Casein,  70 

standard  solution  of,  95 
Caseinogen,  67 
Cheese,  71 
Chlorides  in  blood 

estimation  of,  237 
<  hlorides  in  urine,  134,  137 

detection  of,  137 

estimation  of,  194,  240 
Cholesterin,  120 
Chromic  period,  87 
Cippolina's  test,  162 
Clotting,  sec  coagulation 
Coagulation 

of  blood,  99 

of  milk,  69 

of  proteins  by  alcohol,  10 

of  proteins  by  heat,  6 
Cole's  test 

for  bile  pigments,  251 

for  glucose,  249 
Collagen,  28 

Colour  reactions  of  proteins,  3 
Colorimeter,  191 
Copper-iodide  method 

for  glucose,  227 

for  lactose,  231 
Creatine 

in  muscle,  77 

in  urine,  152 
Creatinine,  78,  152 

estimation,  191 

origin,  152 

properties,  152 

tests,  153; 


Cryoscopy,  126 
Crystallisation 

of  albumin,  16 

of  oxy haemoglobin,  107 
Cyanuric  acid,  140,  144 
Cysteine,  6,  116 
Cystine,  6,  96,  170 

Deposits  in  urine,  169 
Deutero-albumoses,  24 
Dextrins,  45 

detection  of,  204 

formation  of,  45 
Dextrosazone,  38 
Dextrose,  sec  glucose 
Dialysis,  of  serum,  14 
Digestion 

of  carbohydrates,  43,  85 

of  fats,  61 

of  nucleoproteins,  19 

of  proteins  by  pepsin,  24,  88 

of  proteins  by  trypsin,  94 
Disaccharides,  38 
Dubosq's  colorimeter,*491 
Dunstan's  test  for  glycerine,  65 

Earthy  phosphates,  9,  137 
Egg  white,  15 
Egg  albumin,  16 

crystallisation  of,  16 
Emulsification,  60 
Enzymes, 

action  of,  82 

detection  of,  207 

lipase,  63 

pepsin,  88 

ptyalin,  84 

rennin,  70 

trypsin,  94 

Erythro  dextrin,  43,  46 
Esbach's  albuminometer,  199 

reagent,  11 
Estimation  of 

aceto-acetic  acid,  241 

acetone,  241 

acidity  of  gastric  juice,  93 

acidity  of  urine,  129,  193 

albumin, -199 

ammonia,  179-181 

cane  sugar,  58 

chlorides  in  blood,  237 

chlorides  in  urine,  194,  240 

creatinine,  191 

diastase  in  urine,  246 

glucose,  51,  225,  227 

glucose  in  blood,  233 

glycogen,  49 


INDEX. 


255 


lactose,  58,  231 

maltose,  58 

nitrogen,  175-178 

pepsin,  90,  248 

phosphates,  195 

sulphates,  197 

total  nitrogen,  173 

urea,  181-187 

uric  acid,  188-190,  244 
Ethereal  sulphates,  135,  138 

estimation  of,  198 
Euglobulin,  12 

Fats,  59 

digestion  of,  61 

emulsification  of,  60 

in  cheese,  71 

in  milk,  69 

saponification  of,  66 
Fatty  acids,  65 
Fehling's  method,  54 
Fehling's  solution 

preparation  of,  35 
Fehling's  test,  35,  162 
Ferments,  see  enzymes 
Fermentation  test,  163 
Fibrin,  carmine 

preparation  of,  89 
Fibrin  ferment 

formation  of,  100 

preparation  of,  101 
Fibrinogen,  101 
Flour,  72 

Flouride  plasma,  102 
Folin's  fume-absorber,  172 
Folin's  method 

for  acidity  of  urine,  193 

for  ammonia,  179,  180 

for  creatinine,  191 

for  sulphates,  197 

for  total  nitrogen,  175 

for  urea,  183 

for  uric  acid,  189,  244 
Folin-Schaffer  method,  188 
Folin's  test 

for  uric  acid,  149,  150 
Fraunhofer's  lines,  108 
Free  hydrochloric  acid,  91 
Freezing  points,  126 
Fructose,  32,  41,  163 
Fume-absorber,  172 
Furfurol,  40 
Fusion  mixture,  21 

Galactose,  32,  42 
Gelatin,  28 
Gerhardt's  test,  167 


Globulins,  11 

detection  of,  202 

in  muscle,  75 

in  serum,  13 

preparation  of,  13 

properties  of,  11 
Gluconic  acid,  34 
Gluco-proteins,  1,  17 
Glucose,  31 

estimation  of,  51,  57,  225,  227 

fermentation  of,  163 

in  blood,  233 

in  urine,  160,  249 

properties  of,  31-38 

tests  for,  35-38,  249 
Glucosides,  33,  83 
Glucosazone,  38,  162 
Gluten,  72 
Glycerine,  64 

Glycerophosphoric  acid,  122 
Glycocholic  acid,  115 
Glycogen,  48 

estimation  of,  49 

identification  of,  51,  204 

preparation  of,  49 
Glycosuria,  160 
Glycuronic  acid,  34,  167 
Glyoxylic  acid,  4 
Gmelin's  test,  119 
Guanine,  19,  20 
Gunning's  test,  166 
Gunsberg's  reagent,  92 

Haematin,  112 
Haematoporphyrin,  113 

in  urine,  132 
Haematuria,  158 
Haemin,  114,  252 
Haemochromogen,  113 
Haemoglobin,  105,  109 

in  urine,  158 
Haemolysis,  103 
Haser's  coefficient,  125 
Hay's  test,  117 
Heat  coagulation,  6-9 
Heller's  test 

for  albumin,  155 

for  blood,  158 
Hetero  albumose,  24 
Hexoses,  32 
Hippuric  acid,  154 
Histidine,  96 
Histones,  1,  18 
Hopkins'  test 

for  lactic  acid,  80 
Huppert's  test,  119 
Hurtley's  test,  250 


256 


INDEX. 


Hydrochloric  acid 

in  gastric  juice,  91 

normal,  213 

Hydrogen  ions,  concentration  of,  129 
Hydrolysed  proteint,  1,  22 
Hydrolysis 

of  fats,  59,  66 

of  proteins,  24,  96 

of  starch,  43,  45,  85 
Hypobromite, 

action  on  urea,  141,  185 

method,  185 

preparation  of,  142 
Hypoxanthine,  20,  78 

in  urine,  151 

Indican,  168 

Indicators,  131 

Indoxyl,  135,  168 

Inorganic  constituents  of  urine,  133 

Invert  sugar,  39 

lodoform  test,  166 

Iron 

in  haemoglobin,  105 

in  urine,  134 

Jaffe's  test 

for  creatinine,  78,  153 

for  indican,  168 
Jolle's  test,  160 

Katyma's  test,  110 
Keratin,  30 
Kjeldahl's  method,  173 

Lactalbumin,  69 
Lactic  acid,  79 
Lactose,  42 

estimation  of,  58,  231 

in  milk,  69 

in  urine,  164 
Laevulose,  41 

see  also  fructose 
Laking  of  blood,  102 
Larrson's  method 

for  chlorides,  240 
Lavelje's  test,  169 
Lecithin,  122 
Leucine,  96,  98 

Liebermann-Burchard  test,  121 
Ling's  indicator,  55 
Ling's  method,  55 
Lipase 

action  of,  63 

preparation  of,  62 
Logarithms,  see  back  cover 
Long's  coefficient,  125 


Maltodextrine,  43,  46 
Maltose,  41 

estimation  of,  58 
Meat,  see  muscle 
Metaproteins,  22 

detection  of,  202,  203 
Methaemoglobin,  111 
Mett's  tubes,  90 

Microchemical  methods,  175, 233, 237 
Milk,  66 

clotting  of,  69 
Milk  sugar,  see  lactose 
Millon's  reaction,  3 
Molisch's  test,  6 
Monosaccharides,  31 
Moore's  test,  35 
Mucic  acid,  165 
Mucin,  17,  18 

detection  of,  203 

in  bile,  120 

preparation  of,  18 
Mucoid,  16 
Mulder's  test,  37 
Murexide  test,  148 
Muscle,  74 

extract,  75 
Mutarotation,  32 
Myosin,  76 
Myosinogen,  74 

Nessler's  solution,  178 

Neutral  sulphur,  135 

Nitrate  of  urea,  141 

Nitric    acid,     action     on     proteins, 

3,    10,   155 

Nitrogen  in  urine,  estimation  of,  173 
Normal  saline,  103 
Normal  solutions,  preparation  of,  213 
Nucleases,  19 
Nucleic  acid,  19 
Nucleohistone,  18 
Nucleoproteins,  18 

detection  of,  203 

in  bile,  120 

preparation  of,  19 
Nucleosides,  18 
Nucleotides,  18 
Nylander's  test,  37,  161 

Obermayer's  reagent,  169 

Oleic  acid,  59,  65 

Oliver's  test  for  bile  salts,  118,  160 

Osazone 

of  glucose,  38,  162 

of  lactose,  42 

of  maltose,  41 

preparation  of,  38,  162 


INDEX. 


257 


Osmotic  pressure,  126 
Ovo-mucin,  15 
Ovo-mucoid,  16 
•Oxalate 

of  calcium,  170 

of  urea,  141 
Oxalate  plasma,  102 
Oxy-butyric  acid,  166 
Oxy-haemoglobin,  105 

crystallisation  of,  107 

in  urine,  158 

spectrum  of,  108 


Palmitin,  59 
Pancreas,  extract  of,  94 
Parabanic  acid,  145 
Paramyosinogen,  75 
Pentoses,  18,  31 

in  urine,  163 
Pepsin,  88 

action  on  proteins,  24 

detection  of,  89 

estimation  of,  90,  248 
Peptones,  25 

detection  of,  264 

formation  of,  24 

reactions  of,  27 

removals  of,  from  fluids,  27 
Peter's  method  for  sugar,  227 
Pettenkofer's  test,  116 
Phenyl  glucosazone,  38 
Phenyl  hydrazine,  38 
Phenyl  lactosazone,  42 
Phenyl  maltosazone,  41 
Phosphates 

acid,  138 

calcium,  69,  136 

distinction  from  proteins,  202 

earthy,  136,  202 

estimation  of,  195 

in  milk,  69 

in  urine,  136 

stellar,  170 

triple,  170 

Phosphoproteins,  1,  67 
Phosphorus  in  proteins,  21,  69 
Pigments,  identification  of,  206 

of  bile,  118,  251 

of  blood>  105 

of  muscle,  174 

of  urine,  131 
Piotrowski's  reaction,  5 
Plasma,  100 

fluoride,  102 

oxalate,  102 

salted,  101 


Polypeptides,  5 
Polysaccharides,  42 
Potatoes,  71 
Primary  albumoses,  24 
Proline,  97 
Proteins,  1-30 

classification  of,  1 

colour  reactions  of,  3 

crystallisation  of,  16 

detection  of,  202 

hydrolysis  of,  96 

in  bile,  120 

in  urine,  155 

of  muscle,  74 

of  plasma,  101 

of  serum,  6 

peptic  digestion  of,  24,  < 

phosphorus  in,  21,  69 

properties  of,  2 

solubilities  of,  2 

sulphur  in,  6 

tryptic  digestion  of,  94 
Proteoses,  see  albumoses 
Prothrombin,  99 
Proto-albumose,  24 
Prout-Winter  method,  93 
Pseudo-globulin,  12 
Pseudo-mucin,  120 
Ptyalin,  action  of,  85 
Purine  bases,  19,  20 

in  meat,  78 

in  urine,  151 
Purpuric  acid,  149 
Pyrimidine  bases,  19 


Reduced  alkaline  haematin,  113 
Reduced  oxalic  acid,  4 
Reducing  sugars,  34,  161 
Removal  of  proteins,  15,  27 
Rennet  ferment,  70 
Roberts'  test,  156 
Rochelle  salt,  36 
Rothera's  test,  166,  250 


Saccharic  acid,  34 
Saccharose,  see  cane  sugar 
Safranine  test,  37 
Saliva,  84 
Salkowski  test 

for  cholesterin,  121 

for  creatinine,  153 
Salted  plasma,  101 
Saponification,  66 
Sarcolactic  acid,  79 
Sarcosine,  77 


258 


INDEX. 


Scherer's  method,  199 
Schiff  s  test,  149 
Schumm's  test,  158 
Scott-Wilson's  method,  241 
Secondary  albumoses,  25 
Sediments  in  urine,  169 
Seiiwanoff's  test,  41,  163 
Serum,  6,  100 
Soaps,  65 

formation  of,  66 
Solids,  analysis  of,  209 
Soluble  myosin,  75 
Soluble  starch,  45 
Specific  gravity    - 
of  milk,  68 
of  urine,  124 

Specific  oxygen  capacity,  106 
Spectroscope,  107 
Spiegler's  test,  156 
Standard  acids,  213 
Starch,  42 

digestion  of,  ^'3,  85 
grains,  42,  44 
hydrolysis  of,  43 
paste,  44 
reactions  of,  44 
soluble,  45 
Steapsin,  see  lipase 
Stearin,  59 

Stellar  phosphates,  170 
Stercobilin,  119 
Stereoisomerism,  32,  79 
Stokes'  fluid,  109 
Sucrose,  39 
Sugars,  31 

estimation  of,  51,  225,  233 
in  urine,  161,  249 
reducing,  34 
Sulphates  in  urine,  134 

estimation  of,  197 
Sulphur 

estimation  of,  198 
in  proteins,  6 
in  urine,  135 
Sulphur  test 
for  bile  salts,  117 

Taurine,  116 

Teichmann's  crystals,  114,  252 
Temperature  indicator.  184 
Tenison  of  aqueous  vapour,  212 
Thrombin,  99 

preparation  of,  101 
Thrombokinase,  99 
Tollen's  test 

for  glycurbnic  acid,  167 

for  pentoses,  164 


|       Total  nitrogen 

estimation  of,  173-175 

Triple  phosphates,  170 

Trommer's  test,  35 

Trypsin,  94 

detection  of,  95 
preparation  of,  94 
products  of  action,  95,  97 

Tryptophane,  5,  96,  97,  98 

Tyrosine,  4,  96 


Uffelmann's  test,  80 
Urates,  145,  170 
Urea,  139 

detection  of,  205 

estimation  of,  181-187 

in  urine,  144 

nitrate,  141 

oxalate,  141 
Uric  acid,  144 

crystals  of,  145,  148,  169 

estimation  of,  188-190,  244 

in  urine,  150 

origin  of,  20 
Uricase,  20 
Urine 

abnormal,  155 

acidity  of,  129,  193 

albumin  in,  155 

average  composition  of,  123 

deposit  in,  169 

diastase  in,  246 

inorganic  constituents  of,  133 

pigments  of,  131 

proteins  in,  155 

specific  gravity  of,  124 

sugar  in,  160,  249 

total  nitrogen  of,  173 

total  solids  of,  125 
Urinometer,  125 
Urobilin,  119,  132 
Urochrome,  131 
Urcerythrin,  132 
Urorosein,  132 

Volhard's  method,  194 

Weights  and  Measures,  211 

Weyl's  test,  78 

Wheat  flour,  72 

Whey,  70 

Witte's  peptone,  23 

Wohlgemuth's  method,  246 

Xanthine,  20,  74 
Xanthoproteic  test,  3 


14  DAY  USE 

RETURN  TO  DESK  FROM  WHICH  BORROWED 

j»|j;'  •'/         fl      'IPf^^PV 

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This  book  is  due  on  the  last  date  stamped  below,  or 

on  the  date  to  which  renewed. 
Renewed  books  are  subject  to  immediate  recall. 


OCT  2  7  1961 


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